Formation Of Osteoclast-like Multinucleated Cells Research Articles

Melittin inhibits osteoclast formation through the downregulation of the RANKL-RANK signaling pathway and the inhibition of interleukin-1β in murine macrophages.

Melittin is a major toxic component of bee venom (Apis mellifera). It is not known whether melittin is involved in bone metabolism and osteoclastogenesis. The aim of this study was to determine the role of melittin in the regulation of osteoclastogenesis. In vitro osteoclastogenesis assays were performed using mouse RAW 264.7 cells and bone marrow-derived macrophages (BMMs) treated with receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Morphologic and functional analyses for osteoclast-like multinucleated cells (MNCs) were performed by tartrate-resistant acid phosphatase (TRAP) staining, F-actin staining and pit formation methods. The gene expression of TRAP, cathepsin K, matrix metalloproteinase-9 (MMP-9) and carbonic anhydrase II was measured by reverse transcription-quantitative PCR. The protein expression levels of mitogen-activated protein kinases (MAPKs), the p65 subunit of nuclear factor-κB (NF-κB), c-Fos, c-Jun, nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), TNF receptor-associated factor-6 (TRAF6), and interleukin-1β (IL-1β) were assessed by western blot analysis. Melittin inhibited the mRNA expression of TRAP, cathepsin K, MMP-9 and carbonic anhydrase II in RANKL-stimulated RAW 264.7 cells. The increased protein expression of TRAF6, p-extracellular signal-regulated kinase (ERK), p-JNK, p-p65, p-c-Fos and NFATc1 induced by RANKL was significantly suppressed in the RAW 264.7 cells treated with melittin. A synergistic effect of IL-1β on the formation of RANKL-induced osteoclast-like MNCs was found in two experimental cells. The increased expression of IL-1β following the stimulation of RAW 264.7 cells with RANKL activated TRAF6, p-ERK, p-JNK, p-p65, p-c-Fos and NFATc1. These effects were attenuated by the downregulation of IL-1β using siRNA against IL-1β, and also by treatment with melittin. On the whole, the findings of this study demonstrate that melittin inhibits the formation of osteoclast-like MNCs by interfering with the RANKL-RANK signaling pathway.

Tumor-expressed adrenomedullin accelerates breast cancer bone metastasis.

IntroductionAdrenomedullin (AM) is secreted by breast cancer cells and increased by hypoxia. It is a multifunctional peptide that stimulates angiogenesis and proliferation. The peptide is also a potent paracrine stimulator of osteoblasts and bone formation, suggesting a role in skeletal metastases—a major site of treatment-refractory tumor growth in patients with advanced disease.MethodsThe role of adrenomedullin in bone metastases was tested by stable overexpression in MDA-MB-231 breast cancer cells, which cause osteolytic bone metastases in a standard animal model. Cells with fivefold increased expression of AM were characterized in vitro, inoculated into immunodeficient mice and compared for their ability to form bone metastases versus control subclones. Bone destruction was monitored by X-ray, and tumor burden and osteoclast numbers were determined by quantitative histomorphometry. The effects of AM overexpression on tumor growth and angiogenesis in the mammary fat pad were determined. The effects of AM peptide on osteoclast-like multinucleated cell formation were tested in vitro. A small-molecule AM antagonist was tested for its effects on AM-stimulated ex vivo bone cell cultures and co-cultures with tumor cells, where responses of tumor and bone were distinguished by species-specific real-time PCR.ResultsOverexpression of AM mRNA did not alter cell proliferation in vitro, expression of tumor-secreted factors or cell cycle progression. AM-overexpressing cells caused osteolytic bone metastases to develop more rapidly, which was accompanied by decreased survival. In the mammary fat pad, tumors grew more rapidly with unchanged blood vessel formation. Tumor growth in the bone was also more rapid, and osteoclasts were increased. AM peptide potently stimulated bone cultures ex vivo; responses that were blocked by small-molecule adrenomedullin antagonists in the absence of cellular toxicity. Antagonist treatment dramatically suppressed tumor growth in bone and decreased markers of osteoclast activity.ConclusionsThe results identify AM as a target for therapeutic intervention against bone metastases. Adrenomedullin potentiates osteolytic responses in bone to metastatic breast cancer cells. Small-molecule antagonists can effectively block bone-mediated responses to tumor-secreted adrenomedullin, and such agents warrant development for testing in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0458-y) contains supplementary material, which is available to authorized users.

Flavanone Glycosides from Viscum coloratum and Their Inhibitory Effects on Osteoclast Formation

Two novel flavanone glycosides, homoeriodictyol 7-O-β-D-[6-(3-hydroxybutanoyl)glucopyranoside] (viscumneoside IX; 1) and homoeriodictyol 7-O-β-D-[6-(3-hydroxybutanoyl)glucopyranosyl](1→2)-β-D-glucopyranoside (viscumneoside X; 2), together with four known flavanoids, 2-homoeriodictyol 7-O-β-D-glucopyranoside (3), viscumneoside I (4), viscumneoside III (5), and 4',5-dihydroxy-3'-methoxy-7-(2-O-α-L-rhamnopyranosyl-β-D-glucopyranosyloxy)flavanone (6) were isolated from stems and leaves of Viscum coloratum. Their structures were elucidated on the basis of their NMR spectra, HR-FAB-MS data, and acid hydrolysis. Inhibitory effects of the four compounds 1-4 on the formation of osteoclast-like multinucleated cells were investigated. As a result, all the four flavanoids showed significant inhibitory effects on the formation of osteoclast-like multinuclear cells even at a low concentration of 2 μg/ml. The activities of 1-4 at such a concentration exceeded or approximated to that of elcitonin, the positive control drug at a concentration of 2 U/ml, suggesting that they may be of interest for the development of new anti-osteoporosis drugs.

Glycoside modification of oleanolic acid derivatives as a novel class of anti-osteoclast formation agents

Oleanolic acid, a natural product, possesses an anti-osteoclast formation activity. Targeting at discovery of novel and potent anti-bone resorption agents, 22 glycosides of oleanolic acid derivatives (including d-galactopyranosides, d-glucopyranosides, d-xylopyranoses, d-arabopyranoses and d-glycuronic acids) were synthesized at phase-transfer-catalyzed conditions (K 2CO 3, Bu 4NBr, CH 2Cl 2–H 2O) and their inhibitory activity on the formation of osteoclast-like multinucleated cells (OCLs) induced by 1α, 25-dihydroxy vitamin D 3 was evaluated in a co-culture assay system. The structure–activity relationships of these compounds were also discussed.

Synthesis and evaluation of a novel series of heterocyclic oleanolic acid derivatives with anti-osteoclast formation activity

Oleanolic acid with anti-bone resorption effect was an active component discovered in a medicinal plant of Achyranthes bidentata. A series of heterocyclic derivatives of oleanolic acid including indole, pyrazine, quinoxaline, quinoline moieties and their natural amino acid amides were synthesized. Their inhibitory activity on the formation of osteoclast-like multinucleated cells (OCLs) and cytotoxicity of the selected derivatives were evaluated. Among the derivatives, compounds 2a and 8a displayed quite a potent activity even at 200 nM. The structure–activity relationships of the derivatives were also discussed.

Chicken Receptor Activator of Nuclear Factor-κB Ligand Induces Formation of Chicken Osteoclasts from Bone Marrow Cells and also Directly Activates Mature Osteoclasts

Receptor activator of nuclear factor-κB ligand (RANKL), which functions as a major determinant of osteoclast differentiation and activation, is a type II transmembrane protein and is expressed in osteoblasts-stromal cells. The aim of this study was to clarify the role of chicken RANKL (chRANKL) in chicken osteoclast differentiation and to determine its effect on mature chicken osteoclasts. In the present study, chRANKL protein was first cloned and expressed in Escherichia coli. We then treated chicken bone marrow cells with chRANKL protein and found that it induced the formation of chicken osteoclast-like multinucleated cells in a dose-dependent manner in the presence of human macrophage colony-stimulating factor. Moreover, the addition of chicken osteoprotegerin could block the effect of chRANKL with regard to osteoclast-like multi-nucleated cell formation and bone resorption. Using primary cultures of chicken osteoclasts on bone slices, we also found that bone resorption pits per cell increased with chRANKL concentration in a dose-dependent manner. The chRANKL-treated hens exhibited increased blood Ca++ levels within 2 h after injection, showing that chRANKL also activates osteoclasts in vivo. These results clearly indicate that the expressed protein is functional and may also be a critical factor for chicken osteoclastogenesis and bone resorption.

Constitutive c-fos expression in osteoblastic MC3T3-E1 cells stimulates osteoclast maturation and osteoclastic bone resorption.

The effect of culture supernatants of c-fos-transfected MC3T3-E1 osteoblastic cells on osteoclastic bone resorption was studied. Human c-fos cDNA was integrated in the expression vector pH8, and the cells were transfected using the calcium phosphate precipitation technique. Osteoclastic bone resorption was quantified by the pit formation assay, and the osteoclast maturation from precursor was assessed by the generation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNC). The culture supernatants of MC3T3-E1 transfectants constitutively expressing c-fos gene enhanced osteoclast-like MNC formation from haematopoietic blast cells compared with those of control transfectants (P < 0.01). The culture supernatants also promoted osteoclastic bone resorption: the pit number, 118.7 +/- 38.5, was significantly higher than 19.0 +/- 10.1 of the control (P < 0.05). The absorption area, 12,394 +/- 3145 mm2, was significantly larger than 1646 +/- 314 mm2 of the control (P < 0.05). The culture supernatants also promoted bone resorption by purified chick osteoclasts (P < 0.05). The results show that constitutive expression of c-fos gene in osteoblastic MC3T3-E1 cells stimulates osteoclast maturation and osteoclastic bone resorption by releasing humoral mediator(s).

Inhibitory Activity of the Ethyl Acetate Fraction from Viscum coloratum on Bone Resorption

The effects of four fractions, a hexane fraction, an ethyl acetate fraction, an N-butanol fraction and a water fraction, from a water extract of Herba Visci were investigated to find the fraction, in IN VITRO experiments, with the greatest ability to inhibit the formation of osteoclast-like multinucleated cells from mouse bone marrow cells and also to inhibit (45)Ca release from a mouse parietal bone organ culture system. The ethyl acetate fraction was able to inhibit the formation of osteoclast-like cells in a dose-dependent manner and was also able to potently inhibit the release of (45)Ca even at a low concentration. Therefore, a further IN VIVO experiment was performed to determine if the ethyl acetate fraction had antiosteoporotic activity. It was found to inhibit the decreases in total and cancellous bone mineral density, in cancellous and cortical bone mineral content as well as in cortical bone thickness and in the X-axis strength index of tibiae of ovariectomized rats. The principle constituents of the ethyl acetate fraction were determined to be (+)-syringaresinol O- beta-glucopyranoside, 2-homoeriodictyol 7- O-glucoside and viscumneoside I by HPLC analysis.

A novel member of the calcitonin gene-related peptide family, calcitonin receptor-stimulating peptide, inhibits the formation and activity of osteoclasts

We isolated a novel peptide, calcitonin receptor-stimulating peptide-1 (CRSP-1), from porcine brain and found that the administration of this peptide into rats induced a transient decrease in plasma calcium concentration. Therefore, we investigated the effects of CRSP-1 on osteoclastogenesis. Osteoclast-like cells were formed from spleen cells or bone marrow cells by a combination of the receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). CRSP-1 dose-dependently inhibited the formation of multinucleated osteoclast-like cells, and a calcitonin receptor inhibitor antagonized in part the inhibition of osteoclast formation by CRSP-1. Furthermore, CRSP-1 destroyed the actin ring that is a typical index of osteoclast resorption activity; it contributed to this action via the signaling pathway of protein kinase A. Our findings indicate that CRSP-1 inhibits osteoclastogenesis by inhibiting the formation and activity of multinucleated osteoclasts. The inhibitory effects of CRSP-1 on osteoclast metabolism were similar in degree to those of porcine calcitonin. CRSP-1 might provide a clue to the development of tools useful in the prevention and treatment of osteoporosis.

Five New Oleanolic Acid Glycosides fromAchyranthes bidentatawith Inhibitory Activity on Osteoclast Formation

Bioassay-directed fractionation of a butanol-soluble fraction of methanol extract of the root of Achyranthes bidentata resulted in the isolation of 5 new oleanolic acid glycosides 1-5, namely, 18-(beta-D-glucopyranosyloxy)-28-oxoolean-12-en-3beta-yl 3-O-(beta-D-glucopyranosyl)-beta-D-glucopyranosiduronic acid methyl ester (1), achyranthoside C dimethyl ester (2), achyranthoside C butyl dimethyl ester (3), achyranthoside E dimethyl ester (4), and achyranthoside E butyl methyl ester (5), together with 10 known compounds. Their structures were established on the basis of spectroscopic interpretation and chemical methods. All the oleanolic acid glycosides inhibited the formation of osteoclast-like multinucleated cells (OCLs) induced by 1alpha,25(OH)2D3 in a co-culture assay system.

Synthesis and activity of oleanolic acid derivatives, a novel class of inhibitors of osteoclast formation

Two series of oleanolic acid derivatives were synthesized and their inhibitory activity on the formation of osteoclast-like multinucleated cells (OCLs) induced by 1α,25-dihydroxy vitamin D 3 was evaluated in a co-culture assay system. The structure–activity relationships, together with electronic structure based on the frontier molecular orbitals, for example, HOMO and LUMO, related to different amino acid substituents were studied. Derivatives with proline or phenylalanine showed a tendency to enhance the inhibitory activity.

(Pre-)osteoclasts induce retraction of osteoblasts before their fusion to osteoclasts.

Precursors of osteoclasts seeded on top of a confluent layer of osteoblasts/bone lining cells induced retraction of the latter cells. The (pre)osteoclasts then migrated in the formed cell-free areas and fused to form osteoclast-like cells. Retraction of the osteoblasts/bone lining cells proved to depend on activity of matrix metalloproteinases, and TGF-beta1 prevented the retraction. It is well known that osteoblasts have a profound effect on (pre)osteoclasts in inducing the formation of bone-resorbing osteoclasts. Whether, on the other hand, (pre)osteoclasts also modulate osteoblast activity is largely unknown. Because osteoblasts/bone lining cells have to retract from the surface before resorption of bone by osteoclasts, we addressed the question of whether (pre)osteoclasts have the capacity to induce such an activity. Rabbit calvarial osteoblasts/bone lining cells or periosteal fibroblasts were cultured until confluency, after which rabbit peripheral blood mononuclear cells (PBMCs) were seeded on top of them. The co-cultures were maintained for up to 15 days in the presence or absence of the cytokines transforming growth factor (TGF)-beta1 and TNF-alpha and selective inhibitors of matrix metalloproteinases and serine proteinases. The formation of cell-free areas and the number of TRACP+ multinucleated osteoclast-like cells were analyzed. In addition, formation of cell-free areas was analyzed in co-cultures of osteoblasts with mature osteoclasts. The seeding of PBMCs on a confluent layer of osteoblasts/bone lining cells resulted in the following sequence of events. (1) A low number of PBMCs strongly attached to osteoblasts. 2) At these sites of contact, the osteoblasts retracted, thus forming cell-free areas. (3) The PBMCs invaded these areas and attached to the surface of the well, after which they fused and formed multinucleated TRACP+ osteoclast-like cells. Retraction was only seen if the cells were in direct contact; conditioned media from cultured PBMCs added to osteoblasts had no effect. Mature osteoclasts seeded on osteoblasts similarly induced retraction, but this retraction occurred at a much faster rate (within 2 days) than the retraction effectuated by the osteoclast precursors (after 8 days in co-culture). Inhibition of matrix metalloproteinase activity, but not of serine proteinases, strongly reduced retraction of the osteoblasts, thus indicating that this type of cell movement depends on the activity of matrix metalloproteinases. A similar inhibitory effect was found with TGF-beta1. TNF-alpha had no effect on osteoblast retraction but enhanced the formation of multinucleated osteoclast-like cells. Addition of PBMCs to confluent layers of periosteal fibroblasts resulted in similar phenomena as observed in co-cultures with osteoblasts. However, the cell-free areas proved to be significantly smaller, and the number of multinucleated cells formed within cell-free areas was three to four times lower. Our results indicate that osteoclast precursors and mature osteoclasts have the capacity to modulate the activity of osteoblasts and that, yet unknown, membrane-bound signaling molecules are essential in inducing retraction of osteoblasts and the subsequent formation of cell-free areas.

Expression of bone morphogenetic protein-2 messenger ribonucleic acid in cholesteatoma fibroblasts.

The aim of the study was to evaluate the role of bone morphogenetic protein-2 (BMP-2) in the pathology of middle ear cholesteatoma. Middle ear cholesteatoma is a chronic inflammatory disease associated with destruction of the temporal bone and marked by increased expression levels of diverse cytokines. Bone remodeling associated with this disease is mainly caused by the action of osteoclasts. It has been shown that BMP-2 expression is inducible by interleukin 1 in synovial fibroblasts and that BMP-2 in combination with interleukin 1alpha is able to stimulate the formation of osteoclast-like multinucleated cells in co-cultures of osteoblast-like cells and hematopoietic cells. By using Northern hybridizations, we examined the messenger ribonucleic acid expression of BMP-2 in keratinocytes and fibroblasts derived from normal external ear canal skin (EACS) and from cholesteatoma, respectively. We show that normal EACS fibroblasts do not express BMP-2, whereas keratinocytes of both EACS and cholesteatoma origin are positive for the BMP-2 transcript. In contrast to EACS fibroblasts, BMP-2 is clearly expressed in cholesteatoma perimatrix fibroblasts. Incubation of normal fibroblasts with cholesteatoma extracts caused the transcription of BMP-2. Interleukin 1alpha, bacterial endotoxin, or bovine keratin, however, were not able to initiate BMP-2 expression in normal fibroblasts. In view of the above data, it is tempting to speculate that BMP-2 expression might play a role in cholesteatoma pathology.

Tumor necrosis factor-α cooperates with receptor activator of nuclear factor κB ligand in generation of osteoclasts in stromal cell-depleted rat bone marrow cell culture

A member of the tumor necrosis factor (TNF) family, receptor activator of nuclear factor κB ligand (RANKL; also known as ODF, OPGL, and TRANCE), plays critical roles in osteoclast differentiation and activation in the presence of macrophage colony-stimulating factor (M-CSF). Recently, TNF-α has also been shown to induce the formation of multinucleated osteoclast-like cells (MNCs) in the presence of M-CSF from mouse macrophages. We demonstrated that mononuclear preosteoclast-like cells (POCs) were formed in the presence of conditioned medium of osteoblastic cells in a rat bone marrow culture depleted of stromal cells. Using this culture system, in this study we examined whether TNF-α affects differentiation into POCs from hematopoietic progenitor cells. Human TNF-α (hTNF-α) markedly stimulated the formation of POCs. Moreover, a concentration as low as 0.005 ng/mL of hTNF-α increased the level of mRNA for calcitonin receptor (CTR) and cathepsin-K of POCs. The POCs induced by hTNF-α formed MNCs, which showed dentine-resorbing activity after coculture with primary osteoblasts. Stimulation was observed after 24 h of treatment with hTNF-α only on day 1 or day 2 of the culture. After 24 h of hTNF-α treatment, expression of the receptor activator of nuclear factor κB (RANK) mRNA was markedly increased. The addition of soluble RANKL (sRANKL) to the preformed POCs efficiently induced MNCs. Interestingly, treatment of bone marrow cells with hTNF-α and sRANKL synergistically augmented the formation of MNCs. This formation was abolished by the addition of human osteoprotegerin (hOPG). These results suggest that cooperation of TNF-α and RANKL is important for osteoclastogenesis.

Osteoclast differentiation is associated with transient upregulation of cyclin‐dependent kinase inhibitors p21WAF1/CIP1and p27KIP1

Osteoclasts, bone-resorbing multinucleated cells, develop from monocyte-macrophage lineage cells in the presence of osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) and macrophage colony-stimulating factor (M-CSF). M-CSF-dependent bone marrow macrophages (M-BMMPhis) from mouse bone marrow cells have been shown to differentiate into osteoclast-like multinucleated cells (OCLs) in the presence of soluble ODF/RANKL (sODF/RANKL) and M-CSF within 3 days. In this study, we found that stimulation of M-BMMPhis with sODF/RANKL induced a transient expression of cyclin-dependent kinase inhibitors (CDK inhibitors) p21(WAF1/CIP1) and p27(KIP1) by 24 h. The CDK inhibitor proteins disappeared by 48 h. Tumor necrosis factor alpha (TNF-alpha), which is reported to stimulate OCL differentiation, stimulated p21(WAF1/CIP1) and p27(KIP1) expression in M-BMMPhis as well. However, M-CSF alone did not stimulate the expression of the two CDK inhibitors. To clarify the role of p21(WAF1/CIP1) and p27(KIP1) in osteoclastogenesis, accumulation of these CDK inhibitors was aborted by antisense oligonucleotides. Treatment with p21(WAF1/CIP1) antisense oligonucleotide alone, or p27(KIP1) antisense oligonucleotide alone, showed a limited inhibitory effect on OCL formation. However, treatment with a mixture of these two antisense oligonucleotides strongly inhibited OCL formation. These results suggest that a combined modulation of the CDK inhibitors p21(WAF1/CIP1) and p27(KIP1) may be involved in osteoclast differentiation induced by ODF/RANKL.

Dental follicle cell-conditioned medium enhances the formation of osteoclast-like multinucleated cells.

An influx of mononuclear cells and the subsequent increase of osteoclasts around tooth germs suggests that the dental follicle (DF) regulates or influences bone resorption required for tooth eruption. In order to study the effects of DF cell products on osteoclast formation during tooth eruption, a conditioned medium (CM) was created in which DF cells were added to mouse bone marrow cultures. Tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like multinucleated cells were formed in the presence of 10 nM 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The CM, dose-dependently, stimulated the formation of TRAP-positive cells in the presence of 1,25(OH)2D3 for 14 days culture. The number of these cells decreased due to degradation in the control culture. A semi-solid methylcellulose assay in the presence of CM showed little expression of colony-stimulating activity. These results suggest that the DF cells of a developing tooth produce factor(s) that enhance osteoclast formation and bone resorption necessary for tooth eruption.

Cytokine production and bone remodeling in patients wearing overdentures on oral implants.

The stability of titanium dental implants is determined by osseointegration. Bone is a dynamic tissue continuously remodeled through resorption and formation, processes controlled by local cytokine production. This study investigated osseotropic cytokine expression in gingival mucosa, in the intraforamina and inferior first molar zones, during rehabilitation with implant-retained overdentures. Specimens were taken from six patients prior to placement of implants in the intraforamina bone; at connection of healing abutments; and 4, 8, and 12 months after prosthetic anchorage. Through semi-quantitative reverse-transcriptase polymerase chain-reaction, the following constitutively expressed cytokines were found at first surgical stage: interleukin-1, -6, and -8; small amounts of interleukin-11; stem cell factor; and transforming growth factor-beta1, -beta2, and -beta3. From the connection of healing abutments to 12 months after prosthetic anchorage, transforming growth factor-beta1, -beta2, and -beta3 were markedly higher than initial values. Expression of interleukin-6 and -8 decreased 8 months after prosthetic anchorage, while that of interleukin-1 increased at 12 months. In cultured gingival fibroblasts, modulation of cytokine secretion was also time-dependent. Cell culture supernatants influenced osteoclast-like multinucleated cell formation in long-term human marrow culture or osteoblast function, depending on the cytokine profile produced. These results are consistent with functional contributions of cytokines to osseointegration and minimization of posterior edentulous zone bone resorption.

The effect of low molecular weight chitosan on bone resorption in vitro and in vivo.

We studied the effect of low molecular weight chitosan (LMWC) on the formation of osteoclast-like multinucleated cells (OCLs) in the co-culture of mouse osteoblastic cells and bone marrow cells in the presence of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. LMWC at 440 microg/ml inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive OCLs induced by 1alpha,25(OH)2D3. We prepared OCLs in the co-culture of osteoblastic cells and bone marrow cells. The effect of LMWC on pit formation by OCLs was examined using dentin slices, and LMWC inhibited pit formation at 440 microg/ml. Oral administration of the LMWC to ovariectomized rats prevented a decrease in bone mineral density (BMD) of the lumbar vertebra without affecting the body and uterus weights. These results suggested that LMWC prevented a decrease in BMD in vivo by inhibiting osteoclastic bone resorption.

Osteoclast-Derived Zinc Finger (OCZF) Protein With POZ Domain, a Possible Transcriptional Repressor, Is Involved in Osteoclastogenesis

AbstractThe differentiation of osteoclasts is regulated by transcription factors expressed in cells of osteoclast lineage. We isolated here a potential transcription factor from a cDNA library of an enriched population of preosteoclasts and osteoclasts. The cDNA encodes a protein with N-terminal POZ domain and C-terminalKrüppel-like zinc fingers. We designate this protein as osteoclast-derived zinc finger (OCZF). OCZF was found to be rat homologue of mouse leukemia/lymphoma-related factor (LRF). Northern blot and in situ hybridization analysis showed OCZF mRNA at a high level in osteoclasts and kidney cells. OCZF had a nuclear targeting sequence and was localized in the nucleus of transfected cells. In addition, OCZF specifically bound to the guanine-rich consensus sequences of Egr-1 and c-Krox. Transient transfection assays indicate that OCZF can repress transcription activity like other POZ domain proteins. Furthermore, antisense but not sense phosphorothioate oligodeoxynucleotides (ODNs) for OCZF cDNA suppressed the formation of osteoclast-like multinucleated cells (MNCs) in bone marrow culture, whereas the same ODNs did not significantly affect the formation of macrophage polykaryons and mononuclear preosteoclast-like cells (POCs). These results suggest that OCZF is a unique transcription factor that plays an important role in the late stage of osteoclastogenesis.

Osteoclast-Derived Zinc Finger (OCZF) Protein With POZ Domain, a Possible Transcriptional Repressor, Is Involved in Osteoclastogenesis

The differentiation of osteoclasts is regulated by transcription factors expressed in cells of osteoclast lineage. We isolated here a potential transcription factor from a cDNA library of an enriched population of preosteoclasts and osteoclasts. The cDNA encodes a protein with N-terminal POZ domain and C-terminalKrüppel-like zinc fingers. We designate this protein as osteoclast-derived zinc finger (OCZF). OCZF was found to be rat homologue of mouse leukemia/lymphoma-related factor (LRF). Northern blot and in situ hybridization analysis showed OCZF mRNA at a high level in osteoclasts and kidney cells. OCZF had a nuclear targeting sequence and was localized in the nucleus of transfected cells. In addition, OCZF specifically bound to the guanine-rich consensus sequences of Egr-1 and c-Krox. Transient transfection assays indicate that OCZF can repress transcription activity like other POZ domain proteins. Furthermore, antisense but not sense phosphorothioate oligodeoxynucleotides (ODNs) for OCZF cDNA suppressed the formation of osteoclast-like multinucleated cells (MNCs) in bone marrow culture, whereas the same ODNs did not significantly affect the formation of macrophage polykaryons and mononuclear preosteoclast-like cells (POCs). These results suggest that OCZF is a unique transcription factor that plays an important role in the late stage of osteoclastogenesis.