[Show abstract][Hide abstract] ABSTRACT: Receptor activator of nuclear factor-kappaB ligand (RANKL), osteoprotegerin (OPG), and macrophage-colony stimulating factor play essential roles in the regulation of osteoclastogenesis. Runx2-deficient (Runx2-/-) mice showed a complete lack of bone formation because of maturational arrest of osteoblasts and disturbed chondrocyte maturation. Further, osteoclasts were absent in these mice, in which OPG and macrophage-colony stimulating factor were normally expressed, but RANKL expression was severely diminished. We investigated the function of Runx2 in osteoclast differentiation. A Runx2-/- calvaria-derived cell line (CA120-4), which expressed OPG strongly but RANKL barely, severely suppressed osteoclast differentiation from normal bone marrow cells in co-cultures. Adenoviral introduction of Runx2 into CA120-4 cells induced RANKL expression, suppressed OPG expression, and restored osteoclast differentiation from normal bone marrow cells, whereas the addition of OPG abolished the osteoclast differentiation induced by Runx2. Addition of soluble RANKL (sRANKL) also restored osteoclast differentiation in co-cultures. Forced expression of sRANKL in Runx2-/- livers increased the number and size of osteoclast-like cells around calcified cartilage, although vascular invasion into the cartilage was superficial because of incomplete osteoclast differentiation. These findings indicate that Runx2 promotes osteoclast differentiation by inducing RANKL and inhibiting OPG. As the introduction of sRANKL was insufficient for osteoclast differentiation in Runx2-/- mice, however, our findings also suggest that additional factor(s) or matrix protein(s), which are induced in terminally differentiated chondrocytes or osteoblasts by Runx2, are required for osteoclastogenesis in early skeletal development.
[Show abstract][Hide abstract] ABSTRACT: Previous studies have reported enhanced osteoclastogenesis, increased bone resorption and osteoporosis in osteoprotegerin (OPG)-deficient mice. In the present study, we show that the tibial epiphyses contain abundant, thin trabeculae lined with numerous osteoclasts and cuboidal osteoblasts. The increase in osteoblasts and osteoclasts was associated with a dramatic increase in calcein labelling of the mineralization fronts and replacement of much of the intertrabecular marrow with numerous alkaline phosphatase-positive preosteoblasts. Furthermore, the discrete, linear cement lines seen in wild-type mice were replaced by a randomly oriented meshwork of cement lines that were stained intensely for tartrate-resistant acid phosphatase and osteopontin in the OPG-/- mice. These indices of accelerated bone remodelling in mutant bone were associated with irregular trabecular surfaces, a disorganized collagen matrix interspersed with amorphous ground substance and numerous fissures between old and new bone. In total, these observations indicate that enhanced osteoclastic activity in OPG-/- epiphyses led to a coupled increase in osteoblast differentiation and activity and an increase in bone remodelling. The high bone turnover, disorganized matrix and impaired attachment of new to old bone in the cement lines in OPG-/- mice appear to cause bone fragility.
Journal of Electron Microscopy 02/2003; 52(6):503-13. DOI:10.1093/jmicro/52.6.503 · 1.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Bone and Mineral Metabolism 02/2002; 20(6):337-44. DOI:10.1007/s007740200049 · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Biochemical and Biophysical Research Communications 11/2001; 288(1):217-24. DOI:10.1006/bbrc.2001.5745 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transforming growth factor β (TGF-β) is abundant in bone and has complex effects on osteolysis, with both positive and negative effects on osteoclast differentiation, suggesting that it acts via more than one mechanism. Osteoclastogenesis is determined primarily by osteoblast (OB) expression of the tumor necrosis factor (TNF)-related molecule receptor activator of NF-κB ligand (RANKL) and its decoy receptor osteoprotegerin (OPG), which are increased and decreased, respectively, by osteolytic factors. A RANKL-independent osteoclastogenic mechanism mediated by TNF-α has also been shown. Therefore, we investigated TGF-β effects on osteoclast formation in culture systems in which osteoclastogenic stimulus is dependent on OBs and culture systems where it was provided by exogenously added RANKL or TNF-α. Both OPG and TGF-β inhibited osteoclast formation in hemopoietic cell/OB cocultures, but the kinetics of their action differed. TGF-β also inhibited osteoclastogenesis in cocultures of cells derived from OPG null (opg−/−) mice. TGF-β strongly decreased RANKL messenger RNA (mRNA) expression in cultured osteoblasts, and addition of exogenous RANKL to TGFβ-inhibited cocultures of opg−/− cells partially restored osteoclastogenesis. Combined, these data indicate that the inhibitory actions of TGF-β were mediated mainly by decreased OB production of RANKL. In contrast, in the absence of OBs, TGF-β greatly increased osteoclast formation in recombinant RANKL- or TNF-α-stimulated cultures of hemopoietic cells or RAW 264.7 macrophage-like cells to levels several-fold greater than attainable by maximal stimulation by RANKL or TNF-α. These data suggest that TGF-β may increase osteoclast formation via action on osteoclast precursors. Therefore, although RANKL (or TNF-α) is essential for osteoclast formation, factors such as TGF-β may powerfully modify these osteoclastogenic stimuli. Such actions may be critical to the control of physiological and pathophysiological osteolysis.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 09/2001; 16(10):1787 - 1794. DOI:10.1359/jbmr.2001.16.10.1787 · 6.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Osteoclast differentiation factor (ODF), a ligand for osteoclastogenesis inhibitory factor (OCIF)/ osteoprotegerin (OPG), is a member of the membrane-associated tumor necrosis factor (TNF) family and induces osteoclast-like cell formation in vitro. In the present study, mouse ODF genomic clones were isolated and sequenced to determine their gene structure. The mouse ODF gene is a single copy gene consisting of five exons and spans approximately 40kb of the mouse genome. The first exon encodes the intracellular and transmembrane domains. The extracellular region of ODF containing the TNF homologous domain is encoded by exons 1 through 5. The translation-termination codon and six polyadenylation signal residues are present in exon 5. A major transcription-initiation site is present 143 nucleotides upstream of the initiation-ATG codon. This genomic organization is similar to that of other members of the TNF family, especially the CD40 ligand.
[Show abstract][Hide abstract] ABSTRACT: Osteoclastogenesis inhibitory factor (OCIF) is a novel soluble-form member of the tumor necrosis factor receptor family and is involved in the regulation of bone mass. Here we isolated genomic and cDNA clones for mouse OCIF and determined their structures. Mouse OCIF gene spans 29 kb and contains five exons of 270, 367, 192, 225 and 1765 bp long. Four cysteine-rich domains and two death domain homologous regions characterized in human OCIF are rigidly conserved in mouse OCIF. The onset of OCIF gene expression in mouse embryogenesis is at day 8.5. In a pregnant female mouse, OCIF gene is expressed in decidua, a maternal tissue surrounding each embryo, immediately after implantation. The isolation of mouse OCIF gene should facilitate studies on OCIF knock-out mice for a better understanding of the role of OCIF in vivo.
[Show abstract][Hide abstract] ABSTRACT: Osteoclasts are multinucleated cells that resorb bone. Osteoclastogenesis inhibitory factor (OCIF), also called osteoprotegerin (OPG), acts as a naturally occurring decoy receptor for osteoclast differentiation factor, which mediates an essential signal to osteoclast progenitors for their differentiation into osteoclasts. Here we show that the OCIF/OPG knockout mice exhibited severe osteoporosis due to enhanced osteoclastogenesis when they grew to be adults. These mice were viable and fertile. They exhibited marked bone loss accompanied by destruction of growth plate and lack of trabecular bone in their femurs. The strength of their bones dramatically decreased. These results demonstrate that OCIF/OPG is a key factor acting as a negative regulator against osteoclastogenesis. The OCIF/OPG knockout mice provide the first animal model for osteoporosis without other obvious abnormalities.
Biochemical and Biophysical Research Communications 07/1998; 247(3):610-5. DOI:10.1006/bbrc.1998.8697 · 2.30 Impact Factor