Intracellular calcium and protein kinase C mediate expression of receptor activator of nuclear factor-kappaB ligand and osteoprotegerin in osteoblasts

Receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) produced by osteoblasts/stromal cells are involved as positive and negative regulators in osteoclast formation. Three independent signals have been proposed to induce RANKL expression in osteoblasts/stromal cells: vitamin D receptor-, cAMP-, and gp130-mediated signals. We previously reported that intracellular calcium-elevating compounds such as ionomycin, cyclopiazonic acid, and thapsigargin induced osteoclast formation in cocultures of mouse bone marrow cells and primary osteoblasts. Increases in calcium concentration in culture medium also induced osteoclast formation in cocultures. Treatment of primary osteoblasts with these compounds or with high calcium medium stimulated the expression of both RANKL and OPG messenger RNAs (mRNAs). 1,2-Bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)-tetra(acetoxymethyl)ester, an intracellular calcium chelator, suppressed both ionomycin-induced osteoclast formation in cocultures and expression of RANKL and OPG mRNAs in primary osteoblasts. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, also stimulated osteoclast formation in these cocultures and the expression of RANKL and OPG mRNAs in primary osteoblasts. Protein kinase C inhibitors such as calphostin and staurosporin suppressed ionomycin- and PMA-induced osteoclast formation in cocultures and expression of RANKL and OPG mRNAs in primary osteoblasts. Ionomycin stimulated RANKL mRNA expression in ST2 and MC3T3-G2/PA6 cells, but not in MC3T3-E1 or NIH-3T3 cells. These effects were closely correlated with osteoclast formation in response to ionomycin in cocultures with these stromal cell lines. OPG strongly inhibited osteoclast formation induced by calcium-elevating compounds and PMA in cocultures, suggesting that RANKL expression in osteoblasts is a rate-limiting step for osteoclast induction. Forskolin, an activator of cAMP signals, also stimulated osteoclast formation in cocultures. Forskolin enhanced RANKL mRNA expression but suppressed OPG mRNA expression in primary osteoblasts. These results suggest that the calcium/protein kinase C signal in osteoblasts/stromal cells is the fourth signal for inducing RANKL mRNA expression, which, in turn, stimulates osteoclast formation.     

Skeletal and extraskeletal actions of denosumab

Osteoclasts and osteoblasts define skeletal mass, structure and strength through their respective actions in resorbing and forming bone. This remodeling process is orchestrated by the actions of hormones and growth factors, which regulate a cytokine system comprising the receptor activator of nuclear factor κB ligand (RANKL), its receptor RANK and the soluble decoy receptor osteoprotegerin (OPG). Bone resorption depends on RANKL, which determines osteoclast formation, activity and survival. Importantly, cells of the osteoblastic lineage mainly provide RANKL and therefore, are central in the regulation of osteoclast functions. Catabolic effects of RANKL are inhibited by OPG, a TNF receptor family member that binds RANKL, thereby preventing the activation of its receptor RANK, which is expressed by osteoclast precursors. Because this cytokine network is pivotal for the regulation of bone mass in health and diseases, including osteoporosis, rheumatoid arthritis and malignant bone conditions, it has been successfully used for the generation of a targeted therapy to block osteoclast actions. The clinical approval of denosumab, a fully monoclonal antibody against RANKL, provides a novel option to treat bone diseases with a potent, targeted and reversible inhibitor of bone resorption. Although RANKL is also expressed by endothelial cells, T lymphocytes, synovial fibroblasts and various tumor cells, no meaningful clinical extraskeletal effects have been reported after administration of denosumab. This article summarizes the molecular and cellular basis of the RANKL/RANK/OPG system and presents preclinical and clinical studies on the skeletal actions of denosumab.     

Cross-talk between the interleukin-6 and prostaglandin E(2) signaling systems results in enhancement of osteoclastogenesis through effects on the osteoprotegerin/receptor activator of nuclear factor-{kappa}B (RANK) ligand/RANK system

The osteoprotegerin (OPG)/receptor activator of nuclear factor-kappaB ligand (RANKL)/receptor activator of nuclear factor-kappaB (RANK) system is the dominant and final mediator of osteoclastogenesis. Abnormalities of this system have been implicated in the pathogenesis of many skeletal diseases. Cyclooxygenase (COX)-2 and prostaglandin (PG)E(2), a major eicosanoid product of the COX-2-catalyzed pathway, play key roles in normal bone tissue remodeling. PGE(2) exerts its actions by binding and activating the E series of prostaglandin (EP) receptor. Activation of EP(2) and EP(4) receptors is associated with PGE(2)-induced osteoclast differentiation. IL-6, a major proinflammatory cytokine, has also been reported to induce osteoclast differentiation. Although interactions between the COX-2/PGE(2) and IL-6 systems have been described in bone cells, the mechanisms underlying these cooperative signaling pathways and the possible involvement of the OPG/RANKL/RANK system have not been fully elucidated. We demonstrate that COX-2, PGE(2), and IL-6 stimulate osteoblast growth and osteoclast differentiation. Effects on osteoclast differentiation, particularly with IL-6, were most marked when osteoclast precursor cells were grown in coculture with osteoblasts, indicating a possible role of the RANK/RANKL/OPG system. COX-2 and PGE(2) stimulated osteoclastogenesis through inhibition of OPG secretion, stimulation of RANKL production by osteoblasts, and up-regulation of RANK expression in osteoclasts. PGE(2) stimulated IL-6 secretion by bone cells, whereas COX-2 inhibitors decreased this same parameter. IL-6, in turn, increased PGE(2) secretion, COX-2, and EP receptor subtype expression in bone cells. Finally, IL-6 was the mediator of PGE(2)-induced suppression of OPG production by osteoblasts. These findings provide evidence for cross-talk between the PGE(2) and IL-6 signaling enhance osteoclast differentiation via effects on the OPG/RANKL/RANK system in bone cells.     

The inhibition of RANKL causes greater suppression of bone resorption and hypercalcemia compared with bisphosphonates in two models of humoral hypercalcemia of malignancy

Humoral hypercalcemia of malignancy (HHM) is mediated primarily by skeletal and renal responses to tumor-derived PTHrP. PTHrP mobilizes calcium from bone by inducing the expression of receptor activator for nuclear factor-kappaB ligand (RANKL), a protein that is essential for osteoclast formation, activation, and survival. RANKL does not influence renal calcium reabsorption, so RANKL inhibition is a rational approach to selectively block, and thereby reveal, the relative contribution of bone calcium to HHM. We used the RANKL inhibitor osteoprotegerin (OPG) to evaluate the role of osteoclast-mediated hypercalcemia in two murine models of HHM. Hypercalcemia was induced either by sc inoculation of syngeneic colon (C-26) adenocarcinoma cells or by sc injection of high-dose recombinant PTHrP (0.5 mg/kg, s.c., twice per day). In both models, OPG (0.2-5 mg/kg) caused rapid reversal of established hypercalcemia, and the speed and duration of hypercalcemia suppression were significantly greater with OPG (5 mg/kg) than with high-dose bisphosphonates (pamidronate or zoledronic acid, 5 mg/kg). OPG also caused greater reductions in osteoclast surface and biochemical markers of bone resorption compared with either bisphosphonate. In both models, hypercalcemia gradually returned despite clear evidence of ongoing suppression of bone resorption by OPG. These data demonstrate that osteoclasts and RANKL are important mediators of HHM, particularly in the early stages of the condition. Aggressive antiresorptive therapy with a RANKL inhibitor therefore might be a rational approach to controlling HHM.     

Osteoprotegerin produced by osteoblasts is an important regulator in osteoclast development and function

Osteoprotegerin (OPG), a soluble decoy receptor for receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoclast differentiation factor, inhibits both differentiation and function of osteoclasts. We previously reported that OPG-deficient mice exhibited severe osteoporosis caused by enhanced osteoclastic bone resorption. In the present study, potential roles of OPG in osteoclast differentiation were examined using a mouse coculture system of calvarial osteoblasts and bone marrow cells prepared from OPG-deficient mice. In the absence of bone-resorbing factors, no osteoclasts were formed in cocultures of wild-type (+/+) or heterozygous (+/-) mouse-derived osteoblasts with bone marrow cells prepared from homozygous (-/-) mice. In contrast, homozygous (-/-) mouse-derived osteoblasts strongly supported osteoclast formation in the cocultures with homozygous (-/-) bone marrow cells, even in the absence of bone-resorbing factors. Addition of OPG to the cocultures with osteoblasts and bone marrow cells derived from homozygous (-/-) mice completely inhibited spontaneously occurring osteoclast formation. Adding 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] to these cocultures significantly enhanced osteoclast differentiation. In addition, bone-resorbing activity in organ cultures of fetal long bones derived from homozygous (-/-) mice was markedly increased, irrespective of the presence and absence of bone-resorbing factors, in comparison with that from wild-type (+/+) mice. Osteoblasts prepared from homozygous (-/-), heterozygous (+/-), and wild-type (+/+) mice constitutively expressed similar levels of RANKL messenger RNA, which were equally increased by the treatment with 1alpha,25(OH)2D3. When homozygous (-/-) mouse-derived osteoblasts and hemopoietic cells were cocultured, but direct contact between them was prevented, no osteoclasts were formed, even in the presence of 1alpha,25(OH)2D3 and macrophage colony-stimulating factor. These findings suggest that OPG produced by osteoblasts/stromal cells is a physiologically important regulator in osteoclast differentiation and function and that RANKL expressed by osteoblasts functions as a membrane-associated form.     

Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families.

Osteoblasts/stromal cells are essentially involved in osteoclast differentiation and function through cell-to-cell contact (Fig. 8). Although many attempts have been made to elucidate the mechanism of the so-called "microenvironment provided by osteoblasts/stromal cells," (5-8) it has remained an open question until OPG and its binding molecule were cloned. The serial discovery of the new members of the TNF receptor-ligand family members has confirmed the idea that osteoclast differentiation and function are regulated by osteoblasts/stromal cells. RANKL, which has also been called ODF, TRANCE, or OPGL, is a member of the TNF ligand family. Expression of RANKL mRNA in osteoblasts/stromal cells is up-regulated by osteotropic factors such as 1 alpha, 25(OH)2D3, PTH, and IL-11. Osteoclast precursors express RANK, a TNF receptor family member, recognize RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into pOCs in the presence of M-CSF. RANKL is also involved in the survival and fusion of pOCs and activation of mature osteoclasts. OPG, which has also been called OCIF or TR1, is a soluble receptor for RANKL and acts as a decoy receptor in the RANK-RANKL signaling system (Fig. 8). In conclusion, osteoblasts/stromal cells are involved in all of the processes of osteoclast development, such as differentiation, survival, fusion, and activation of osteoclasts (Fig. 8). Osteoblasts/stromal cells can now be replaced with RANKL and M-CSF in dealing with the whole life of osteoclasts. RANKL, RANK, and OPG are three key molecules that regulate osteoclast recruitment and function. Further studies on these key molecules will elucidate the molecular mechanism of the regulation of osteoclastic bone resorption. This line of studies will establish new ways to treat several metabolic bone diseases caused by abnormal osteoclast recruitment and functions such as osteopetrosis, osteoporosis, metastatic bone disease, Paget's disease, rheumatoid arthritis, and periodontal bone disease.     

Changing RANKL/OPG mRNA expression in differentiating murine primary osteoblasts

Osteoblast-osteoclast coordination is critical in the maintenance of skeletal integrity. The modulation of osteoclastogenesis by immature cells of the osteoblastic lineage is mediated through receptor activator of NF kappa B (RANK), its ligand RANKL, and osteoprotegerin (OPG), a natural decoy receptor for RANKL. Here, the expression of OPG and RANKL in primary mouse osteoblastic cultures was investigated to determine whether the osteoclastogenic stimulus depended on the stage of osteoblastic differentiation and the presence of the calciotrophic hormone 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)). OPG mRNA expression was increased in osteoblastic cultures after the onset of mineralisation relative to less mature cultures, but did not alter in response to 1,25-(OH)(2)D(3) treatment. In contrast, basal RANK L mRNA expression did not change during differentiation but was significantly enhanced by 1,25-(OH)(2)D(3) treatment at all times. The stimulatory effects of 1,25-(OH)(2)D(3) on RANKL were lessened in more mature cultures, however. The RANKL/OPG ratio, an index of osteoclastogenic stimulus, was therefore increased by 1,25-(OH)(2)D(3) treatment at all stages of osteoblastic differentiation, but to a lesser degree in cultures after the onset of mineralisation. Thus the 1,25-(OH)(2)D(3)-driven increase in osteoclastogenic potential of immature osteoblasts appears to be mediated by increased RANKL mRNA expression, with mature osteoblasts having relatively decreased osteoclastogenic activity due to increased OPG mRNA expression. These findings suggest a possible mechanism for the recently proposed negative regulatory role of mature osteoblasts on osteoclastogenesis and indicate that the relative proportions of immature and mature osteoblasts in the local microenvironment may control the degree of resorption at each specific bone site.     

Osteoblasts and bone formation

Bone is constantly being remodelled in a dynamic process where osteoblasts are responsible for bone formation and osteoclasts for its resorption. Osteoblasts are specialized mesenchymal cells that undergo a process of maturation where genes like core-binding factor alpha1 (Cbfa1) and osterix (Osx) play a very important role. Moreover, it was found recently that Wnt/ beta-catenin pathway plays a part on osteoblast differentiation and proliferation. In fact, mutations on some of the proteins involved in this pathway, like the low-density lipoprotein receptor related protein 5/6 (LRP5/6) lead to bone diseases. Osteoblast have also a role in regulation of bone resorption through receptor activator of nuclear factor-kappaB (RANK) ligand (RANKL), that links to its receptor, RANK, on the surface of pre-osteoblast cells, inducing their differentiation and fusion. On the other hand, osteoblasts secrete a soluble decoy receptor (osteoprotegerin, OPG) that blocks RANK/RANKL interaction by binding to RANKL and, thus, prevents osteoclast differentiation and activation. Therefore, the balance between RANKL and OPG determines the formation and activity of osteoclasts. Another factor that influences bone mass is leptin, a hormone produced by adipocytes that have a dual effect. It can act through the central nervous system and diminish osteoblasts activity, or can have an osteogenic effect by binding directly to its receptors on the surface of osteoblast cells.     

The molecular mechanism of osteoclastic bone resorption and inhibitory drugs for bone resorption

Osteoclasts are the only cells that destroy and resorb bone. The differentiation and activation of osteoclasts are tightly regulated by osteoblasts. Osteoblasts express RANKL essential for osteoclast differentiation. Osteoclast precursors express RANK, a receptor of RANKL, recognized RANKL through cell-cell interaction. Serum levels of RANKL were markedly elevated in deficiency of osteoprotegerin (OPG), a soluble decoy receptor for RANKL. Calcitonin and bisphosphonate are used for inhibitory drugs of bone resorption by osteoclasts. In addition, a specific anti-RANKL monoclonal antibody is expected as well-tolerated bone antiresorptive agent.     

Triiodothyronine (T3) does not induce Rankl expression in rat Ros 17/2.8 cells

Osteoclastogenesis may be regulated via activation of the RANK/RANKL (receptor activator of nuclear factor-kappa B/receptor activator of nuclear factor-kappa B ligand) system, which is mediated by osteoblasts. However, the bone loss mechanism induced by T3 (triiodothyronine) is still controversial. In this study, osteoblastic lineage rat cells (ROS 17/2.8) were treated with T3 (10(-8) M, 10(-9) M, and 10(-10) M), and RANKL mRNA (messenger RNA) expression was measured by semiquantitative RT-PCR. Our results show that T3 concentrations used did not significantly enhance RANKL expression compared to controls without hormone treatment. This data suggests that other mechanisms, unrelated to the RANK/RANKL system, might be to activate osteoclast differentiation in these cells.     

Rank/Rankl/opg: literature review

The discovery of the receptor activator of nuclear factor-kB (RANK)/RANK Ligand (RANKL)/osteoprotegerin (OPG) pathway contributed to the understanding of how bone formation and resorption were processed and regulated. RANKL and OPG are members of the tumor necrosis factor (TNF) and TNF receptor (TNFr) superfamilies, respectively, and binding to receptor activator of NF-kB (RANK) not only regulate osteoclast formation, activation and survival in normal bone modeling and remode-ling, but also in several other pathologic conditions characterized by increased bone turnover. There is accumulating evidence of the potential role of OPG and RANKL in other tissues. Looking beyond the RANK/RANKL/OPG axis, Wingless (Wnt) pathway emerged as the osteoblast differentiation way, and also as a bone mass regulator. Researchers have been discovering new molecules and cytokines interactions. Altogether, data suggest that RANK/RANKL/OPG system could be targeted as a new treatment strategy in bone conditions. FREEDOM is the more recently published clinical trial about a RANKL-specific recombinant fully human monoclonal antibody (denosumab). OPG is also a potential innovative therapeutic option to be investigated.     

Osteoprotegerin inhibits the development of osteolytic bone disease in multiple myeloma.

Multiple myeloma is a B-cell malignancy characterized by the accumulation of plasma cells in the bone marrow and the development of osteolytic bone disease. The present study demonstrates that myeloma cells express the critical osteoclastogenic factor RANKL (the ligand for receptor activator of NF-kappa B). Injection of 5T2MM myeloma cells into C57BL/KaLwRij mice resulted in the development of bone disease characterized by a significant decrease in cancellous bone volume in the tibial and femoral metaphyses, an increase in osteoclast formation, and radiologic evidence of osteolytic bone lesions. Dual-energy x-ray absorptiometry demonstrated a decrease in bone mineral density (BMD) at each of these sites. Treatment of mice with established myeloma with recombinant osteoprotegerin (OPG) protein, the soluble decoy receptor for RANKL, prevented the development of lytic bone lesions. OPG treatment was associated with preservation of cancellous bone volume and inhibition of osteoclast formation. OPG also promoted an increase in femoral, tibial, and vertebral BMD. These data suggest that the RANKL/RANK/OPG system may play a critical role in the development of osteolytic bone disease in multiple myeloma and that targeting this system may have therapeutic potential.     

Receptor activator of nuclear factor kappa B ligand (RANKL): another link between breast and bone.

Mice rendered null for the genes encoding receptor activator of nuclear factor kappa B ligand (RANKL) or its receptor, RANK, are osteopetrotic because of failure of osteoclast development. The failure of lactation owing to the lack of development of lobulo-alveolar structures during pregnancy, despite earlier stages of mammary gland development being normal, is now added to each of these phenotypes. The breast phenotype in RANKL-/- (but not in RANK-/-) mice is rescued by treatment of pregnant mice with RANKL, indicating a key role for these tumour necrosis factor (TNF) ligand and receptor family members in a crucial terminal step in breast development and lactation. Both RANKL and RANK are synthesized by mammary epithelial cells, with both prolactin and parathyroid hormone-related protein (PTHrP) able to enhance production of mRNA for RANKL. These findings reveal a paracrine-autocrine system in lactation control, with novel signalling pathways that reflect intercellular communication processes in bone.     

Osteoclast differentiation factor induces synovial macrophage–osteoclast differentiation in rheumatoid arthritis

The aim of this study was to clarify the role of osteoclast differentiation factor (ODF) and osteoprotegerin (OPG) in synovial macrophage–osteoclast differentiation. Synovial macrophages were cultured in the presence of macrophage-colony-stimulating factor (M-CSF) and/or ODF. OPG was added to cocultures of synovial macrophages and UMR106. The cultures on glass coverslips were stained with osteoclast-associated markers, tartrate-resistant acid phosphatase (TRAP), and vitronectin receptor (VNR), as well as macrophage-associated markers CD11b and CD14. Functional evidence of osteoclast formation was determined by a resorption pit assay. To investigate whether rheumatoid arthritis (RA) synovial cells expressed messenger RNA (mRNA) for ODF, OPG, and the receptor activator of NF-κB (RANK), we performed a polymerase chain reaction (PCR) analysis. The addition of M-CSF or ODF alone induced TRAP-positive multinucleated cell formation. Resorption pits were rarely detected with M-CSF alone. ODF was capable of inducing bone resorption and enhancing osteoclastogenesis, as well as bone resorption in the presence of M-CSF. In the coculture system, both osteoclast formation and bone resorption were inhibited by OPG in a dose-dependent manner. In all experiments, synovial cells, including macrophages and fibroblasts, expressed the mRNA for RANK, ODF, and OPG. Our findings suggest that ODF plays a role in regulating RA synovial macrophage–osteoclast differentiation, and that synovial cells might have the ability to produce ODF. OPG might be further developed as a new strategy for treating bone destruction in RA joints. Received: January 30, 2001 / Accepted: May 18, 2001     

Parathyroid hormone stimulates TRANCE and inhibits osteoprotegerin messenger ribonucleic acid expression in murine bone marrow cultures: correlation with osteoclast-like cell formation.

We studied the effects of PTH on the expression of tumor necrosis factor-related activation-induced cytokine (TRANCE), osteoprotegerin (OPG), and receptor activator of NF kappaB (RANK) messenger RNA (mRNA) in cultured murine bone marrow, calvaria, and osteoblasts. TRANCE, OPG, and RANK are recently identified regulators of osteoclast formation. Bone marrow cells were cultured with or without PTH(1-34) for 6 days. TRANCE, OPG, and RANK mRNA were measured by RT-PCR. In 6-day cultures, PTH stimulated the number of OCL/well in a dose-dependent manner. A time course showed significant (P < 0.01) increases in OCL/well after 24 h of PTH (100 ng/ml). TRANCE mRNA expression, like OCL formation, increased dose dependently and was maximal, with 10-100 ng/ml PTH. In contrast, OPG mRNA expression was decreased by 0.1 ng/ml PTH (40%) and completely abolished by 1 ng/ml. TRANCE mRNA expression was rapidly stimulated by PTH (maximal response at 1 h, 8.1-fold over control). Expression declined by 40% at 24 h but was still much greater than control at 6 days (4.6-fold) in a time-course study. PTH caused a transient stimulation of OPG mRNA at 1 h (2-fold), which returned to basal levels by 2 h. After 6 h, PTH completely inhibited OPG mRNA. There were only minor effects of PTH on RANK mRNA expression. PTH had less potent effects on TRANCE and OPG mRNA expression in calvaria organ cultures and osteoblasts. In mouse calvaria cultures, TRANCE expression was detectable in controls and was increased 2.9-fold by PTH at 24 h. PTH treatment of calvaria decreased OPG expression by 30% at 6 h. MC3T3 E-1 osteoblastic cells expressed minimal levels of TRANCE mRNA either before or after PTH treatment. OPG mRNA was present in MC3T3 E-1 cells, but levels were not modulated by PTH. In primary osteoblastic cells, PTH stimulated TRANCE mRNA expression 4-fold at 2 h and inhibited OPG mRNA expression by 46%. These results demonstrate a tight correlation between the ability of PTH to stimulate OCL formation in marrow culture and expression of TRANCE (r = 0.87, P < or = 0.05) and OPG mRNA (r = -0.88, P < or = 0.05). Reciprocal regulation of TRANCE and OPG mRNA by PTH preceded its effects on OCL formation by 18-23 h. Hence, it is likely that PTH regulates bone resorption, at least in part, via its effects on TRANCE and OPG expression.     

Osteoclast differentiation factor induces synovial macrophage–osteoclast differentiation in rheumatoid arthritis

Abstract

The aim of this study was to clarify the role of osteoclast differentiation factor (ODF) and osteoprotegerin (OPG) in synovial macrophage–osteoclast differentiation. Synovial macrophages were cultured in the presence of macrophage-colony-stimulating factor (M-CSF) and/or ODF. OPG was added to cocultures of synovial macrophages and UMR106. The cultures on glass coverslips were stained with osteoclast-associated markers, tartrate-resistant acid phosphatase (TRAP), and vitronectin receptor (VNR), as well as macrophage-associated markers CD11b and CD14. Functional evidence of osteoclast formation was determined by a resorption pit assay. To investigate whether rheumatoid arthritis (RA) synovial cells expressed messenger RNA (mRNA) for ODF, OPG, and the receptor activator of NF-κB (RANK), we performed a polymerase chain reaction (PCR) analysis. The addition of M-CSF or ODF alone induced TRAP-positive multinucleated cell formation. Resorption pits were rarely detected with M-CSF alone. ODF was capable of inducing bone resorption and enhancing osteoclastogenesis, as well as bone resorption in the presence of M-CSF. In the coculture system, both osteoclast formation and bone resorption were inhibited by OPG in a dose-dependent manner. In all experiments, synovial cells, including macrophages and fibroblasts, expressed the mRNA for RANK, ODF, and OPG. Our findings suggest that ODF plays a role in regulating RA synovial macrophage–osteoclast differentiation, and that synovial cells might have the ability to produce ODF. OPG might be further developed as a new strategy for treating bone destruction in RA joints.     

Osteoblasts provide a suitable microenvironment for the action of receptor activator of nuclear factor-kappaB ligand

Deficiency of osteoprotegerin (OPG), a soluble decoy receptor for receptor activator of nuclear factor-kappaB ligand (RANKL), in mice induces osteoporosis caused by enhanced bone resorption. Serum concentrations of RANKL are extremely high in OPG-deficient (OPG(-/-)) mice, suggesting that circulating RANKL is involved in osteoclastogenesis. RANKL(-/-) mice exhibit osteopetrosis, with the absence of osteoclasts. We examined the requirements for osteoclastogenesis using OPG(-/-) mice, RANKL(-/-) mice, and a system involving bone morphogenetic protein 2 (BMP-2)-induced ectopic bone formation. When collagen disks containing BMP-2 (BMP-2-disks) or vehicle were implanted into OPG(-/-) mice, osteoclast-like cells (OCLs) and alkaline phosphatase-positive OCLs appeared in BMP-2-disks but not the control disks. F4/80-positive osteoclast precursors were similarly distributed in both BMP-2- and control disks. Cells expressing RANKL were detected in the BMP-2-disks, and the addition of OPG to the disk inhibited OCL formation. Muscle cells in culture differentiated into alkaline phosphatase-positive cells in the presence of BMP-2 and accordingly expressed RANKL mRNA in response to PTH. This suggests that RANKL expressed by osteoblasts is a requirement for osteoclastogenesis. We then examined how osteoblasts are involved in osteoclastogenesis other than RANKL expression, using RANKL(-/-) mice. BMP-2- and control disks were implanted into RANKL(-/-) mice, which were injected with RANKL for 7 d. Many OCLs were observed in the BMP-2-disks and bone tissues but not the control disks. These results suggest that osteoblasts also play important roles in osteoclastogenesis through offering the critical microenvironment for the action of RANKL.