Osteoclast-like Cell Formation Research Articles

Inhibitory activities of plastoquinones and chromene derivative from a brown alga Sagassum micracanthum on bone resorption.

We evaluated inhibitory effects of plastoquinones (1, 2) and chromene derivative (3) from the methanol extract of Sargassum micracanthum on the differentiation of osteoclast progenitors into osteoclast-like cells (OCLs), bone-resorbing activity and the survival of OCLs. When OCL formation was investigated using osteoclast progenitor cells obtained from macrophage-colony stimulating factor (M-CSF)-treated mouse bone marrow, 1-3 inhibited dose-dependently OCL formation at the concentrations of 3-10 muM, the order of inhibitory potency being 1>3>2. In addition, they suppressed dose-dependently the pit formation induced by OCL on dentine slices, the order of inhibitory potency being 1>3>2. The survival of OCLs was inhibited by about 35, 45 and 60% in the presence of 6 muM of 2, 3 and 1, respectively. These results suggest that the inhibitory effects of the three compounds on the differentiation, pit formation and the survival of OCLs might contribute to the suppression of bone resorption.

Royal jelly prevents osteoporosis in rats: beneficial effects in ovariectomy model and in bone tissue culture model.

Royal jelly (RJ) has been used worldwide for many years as medical products, health foods and cosmetics. Since RJ contains testosterone and has steroid hormone-type activities, we hypothesized that it may have beneficial effects on osteoporosis. We used both an ovariectomized rat model and a tissue culture model. Rats were divided into eight groups as follows: sham-operated (Sham), ovariectomized (OVX), OVX given 0.5% (w/w) raw RJ, OVX given 2.0% (w/w) RJ, OVX given 0.5% (w/w) protease-treated RJ (pRJ), OVX given 2.0% (w/w) pRJ, OVX given 17β-estradiol and OVX given its vehicle, respectively. The Ovariectomy decreased tibial bone mineral density (BMD) by 24%. Administration of 17β-estradiol to OVX rats recovered the tibial BMD decrease by 100%. Administration of 2.0% (w/w) RJ and 0.5–2.0% (w/w) pRJ to OVX rats recovered it by 85% or more. These results indicate that both RJ and pRJ are almost as effective as 17β-estradiol in preventing the development of bone loss induced by ovariectomy in rats. In tissue culture models, both RJ and pRJ increased calcium contents in femoral-diaphyseal and femoral-metaphyseal tissue cultures obtained from normal male rats. However, in a mouse marrow culture model, they neither inhibited the parathyroid hormone (PTH)-induced calcium loss nor affected the formation of osteoclast-like cells induced by PTH in mouse marrow culture system. Therefore, our results suggest that both RJ and pRJ may prevent osteoporosis by enhancing intestinal calcium absorption, but not by directly antagonizing the action of PTH.

Activated platelets retain their potential to induce osteoclast-like cell formation in murine bone marrow cultures

Supernatants immediately obtained after platelet activation can induce osteoclast-like cell formation in murine bone marrow cultures. Here we report that activated platelets retain their potential to induce osteoclast-like cell formation over a 3-day period with repeated washing, when co-cultured with murine bone marrow cells. Supernatants obtained from washed platelets 3 days following their activation with thrombin, caused the differentiation of haematopoietic progenitors into osteoclast-like cells. The platelet-derived soluble factor(s) responsible for the induction of osteoclastogenesis can be retained in an ultrafilter with a nominal molecular weight limit of 10 kDa, and loose their activity when incubated at 99°C. Indomethacin, which inhibits cyclooxygenase activity, and osteoprotegerin, a decoy receptor for receptor activator of nuclear factor-κB ligand (RANKL), suppressed the formation of osteoclast-like cells in this model. The in vitro findings presented here suggest that activated platelets can induce osteoclast-like cell formation via a prostaglandin and RANKL-dependent mechanism over a time period corresponding to the existence of a blood clot.

RANKL-stimulated osteoclast-like cell formation in vitro is partially dependent on endogenous interleukin-1 production

Receptor activator of NF-κB ligand (RANKL) and interleukin-1 (IL-1) individually plays a critical role in the differentiation and activation of osteoclasts in bone. In addition, both RANKL and IL-1 activate similar signal transduction pathways including p38 MAP kinase and c-Jun NH 2 terminal kinase (JNK). We examined if endogenously produced IL-1 influenced osteoclast-like cell (OCL) formation in murine bone marrow and bone marrow monocyte (BMM) cultures that were stimulated with M-CSF and RANKL. RANKL stimulated OCL formation in a dose-dependent manner in bone marrow cultures, and this response was significantly inhibited by IL-1 RA (100 ng/ml), a specific IL-1 antagonist. Interleukin-1 further increased OCL formation in BMM cultures that were treated with M-CSF (30 ng/ml) and RANKL (1, 3, 10 and 30 ng/ml). In addition, BMM cultures from IL-1 type I receptor-deficient mice, which do not respond to IL-1, demonstrated significantly less OCL formation compared to wild-type BMM cultures. We examined the time course and dose response of IL-1alpha protein expression by ELISA in BMM cultures that were treated with or without M-CSF and RANKL. RANKL dose dependently stimulated IL-1α protein significantly (up to 46%) in 6-day cultures. The interaction of RANKL and IL-1 on osteoclastogenesis did not appear significantly dependent on prostaglandin synthesis since PGE 2 expression in the conditioned medium of BMM cultures was nearly undetectable and the PGHS-2 specific inhibitor, NS-398, was without effect. We also investigated the effect of IL-1 on p38 MAP kinase and JNK in BMM cultures. The combination of RANKL and IL-1 had additive effects on JNK but not p38 MAP kinase compared to results in cultures treated with RANKL or IL-1 alone. In addition, SP600125, a specific JNK inhibitor, markedly reduced OCL formation in BMM cultures that were treated with RANKL or the combination of RANKL and IL-1. These findings demonstrate that endogenously produced IL-1 augments the response of bone marrow cells to RANKL, and this effect appears mediated by mechanisms that are associated with enhancement of JNK activity.

Nicotine and lipopolysaccharide stimulate the formation of osteoclast-like cells by increasing macrophage colony-stimulating factor and prostaglandin E 2 production by osteoblasts

Several studies have indicated that one of the causes of alveolar bone destruction with periodontitis is lipopolysaccharide (LPS) from the cell wall of Gram-negative bacteria in plaque and that tobacco smoking may be an important risk factor for the development and severity of periodontitis. The present study was undertaken to determine the effect of nicotine and LPS on the expression of macrophage colony-stimulating factor (M-CSF), osteoprotegerin (OPG), and prostaglandin E 2 (PGE 2) in osteoblasts, and the indirect effect of nicotine and LPS on the formation of osteoclast-like cells. Saos-2 cells were cultured with 10 − 3 M nicotine, or 1 or 10 μg/ml LPS and 10 − 3 M nicotine, for up to 14 days. The gene and protein expression of M-CSF and OPG were determined using real-time PCR and ELISA, respectively. PGE 2 expression was determined using ELISA. The formation of osteoclast-like cells was estimated using tartrate-resistant acid phosphatase (TRAP) staining of osteoclast precursors in culture with conditioned medium from nicotine and LPS-treated Saos-2 cells and the soluble receptor activator of NF-κB ligand (RANKL). M-CSF and PGE 2 expression increased markedly in cells cultured with nicotine and LPS compared with those cultured with nicotine alone. OPG expression increased in the initial stages of culture with nicotine and LPS but decreased in the later stages of culture. The conditioned medium containing M-CSF and PGE 2 produced by nicotine and LPS-treated Saos-2 cells with soluble RANKL increased the TRAP staining of osteoclast precursors compared with that produced by nicotine treatment alone. These results suggest that nicotine and LPS stimulate the formation of osteoclast-like cells via an increase in M-CSF and PGE 2 production and that the stimulation is greater than with nicotine treatment alone.

Vaccinia Virus Complement Control Protein Ameliorates Collagen‐Induced Arthritic Mice

The main objective of this study was to investigate the therapeutic efficiency of recombinant vaccinia virus complement control protein (rVCP) on collagen-induced arthritis (CIA) in DBA-1/J mice. Arthritis was induced in DBA-1J mice by injecting bovine collagen emulsified in complete Freunds adjuvant. We used rVCP to block complement activation and investigated its effect on different aspects of CIA including osteoclast formation and bone destruction. The osteoclast-like cells were detected using immunohistochemistry. Joint destruction was studied using X-ray of the intact knee joints. Cartilage destruction was monitored by staining the paraffin sections with toluidine blue. ELISA was used to measure the cytokine levels in the serum. Blocking complement activation in DBA/1J arthritic mice with rVCP resulted in significant inhibition of the clinical progression of the disease and reduction in joint destruction as revealed by X-ray analysis and toluidine blue staining of the joint sections. Inhibition of complement reduced the production of proinflammatory cytokines and the number of osteoclast-like cells in arthritic joints. In conclusion, blocking of complement in CIA by rVCP inhibits the inflammation and the formation of osteoclast-like cells and reduces cartilage destruction.

Osteoblast lineage properties in giant cell tumors of bone

BackgroundGiant cell tumors of bone (GCTs), among the most common primary bone tumors, are characterized by the formation of abundant osteoclast-like multinucleated giant cells (MNCs). It is not yet clear about the origin of GCTs and which cells in the lesion are the true neoplastic component. Several recent reports suggested that MNCs are osteoclasts induced by stroma-like tumor cells expressing the ligand for receptor activator of NF-kB (RANKL), which is a membrane-bound osteoclast differentiation factor. This hypothesis suggests an osteoblast lineage origin of GCTs, although it has long been speculated about GCTs being of mesenchymal stem cell (MSC) origin. MethodsWe investigated the expression of osteoblastic differentiation markers in 10 human GCTs by reverse transcription-polymerase chain reaction and immunohistochemistry. We also performed osteoblastic and adipogenic differentiation assays using cultured cells derived from surgically resected lesions to estimate the stem cell-like properties. ResultsGCTs and derived stromal cells expressed many osteoblast lineage marker genes, such as collagen type I, bone sialoprotein, core binding factor a-1, and osteocalcin. Instead of stable expression of mRNA, osteocalcin was not detected among the proteins. The tumor-derived cultures showed osteoblastic but not adipogenic differentiation capability. These findings strongly suggest that GCTs are of osteoblast lineage origin. ConclusionsOur results indicated that GCTs expressed many osteoblastic markers and showed properties of preosteoblast-like cells rather than those of MSCs. These observations may provide some insight into the mechanisms of disease progression and the origin of GCTs.

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.

Mechanisms Involved in Enhancement of Osteoclast Formation and Function by Low Molecular Weight Hyaluronic Acid

Hyaluronic acid (HA) is a component of the extracellular matrix that has been shown to play an important role in bone formation, resorption, and mineralization both in vivo and in vitro. We examined the effects of HA at several molecular weights on osteoclast formation and function induced by RANKL (receptor activator of NF-kappa B ligand) in a mouse monocyte cell line (RAW 264.7). HA at M(r) < 8,000 (low molecular weight HA (LMW-HA)) enhanced tartrate-resistant acid phosphatase-positive multinucleated cell formation and tartrate-resistant acid phosphatase activity induced by RANKL in a dose-dependent manner, whereas HA at M(r) > 900,000 (high molecular weight HA (HMW-HA)) showed no effect on osteoclast differentiation. LMW-HA enhanced pit formation induced by RAW 264.7 cells, whereas HMW-HA did not, and LMW-HA stimulated the expression of RANK (receptor activator of NF-kappa B) protein in RAW 264.7 cells. In addition, we found that LMW-HA enhanced the levels of c-Src protein and phosphorylation of ERKs and p38 MAPK in RAW 264.7 cells stimulated with RANKL, whereas the p38 MAPK inhibitor SB203580 inhibited RANKL-induced osteoclast differentiation. This enhancement of c-Src and RANK proteins induced by LMW-HA was inhibited by CD44 function-blocking monoclonal antibody. These results indicate that LMW-HA plays an important role in osteoclast differentiation and function through the interaction of RANKL and RANK.

Papillary Urothelial Bladder Carcinoma Associated with Osteoclast-Like Giant Cells

We report the case of a papillary urothelial carcinoma associated with osteoclast-like giant cells. A 60-year old woman presented with hematuria. A papillary neoplasm was detected by cystoscopy and removed transurethrally. Histological examination revealed a papillary urothelial carcinoma (grade I) associated with multiple stromal giant cells, which displayed morphological, ultrastructural and immunohistochemical characteristics of osteoclast-like giant cells. The formation of osteoclast-like giant cells in association with urothelial bladder carcinoma is a rare event, of which only six cases have been reported in the Anglo-American literature. It may cause diagnostic problems because primary giant cell tumor, giant cell carcinoma and foreign body stromal reaction have to be considered. Immunohistochemistry and electron microscopy may help to rule out these differential diagnoses.

IL-1α stimulates the formation of osteoclast-like cells by increasing M-CSF and PGE 2 production and decreasing OPG production by osteoblasts

Interleukin-1α (IL-1α) is one of the most potent bone-resorbing factors involved in the bone loss that is associated with inflammation. We examined the effect of the inflammatory mediator IL-1α on the expression of macrophage colony-stimulating factor (M-CSF), osteoprotegerin (OPG), and prostaglandin E 2 (PGE 2) in rat osteoblasts, and the indirect effect of IL-1α on the formation of osteoclast-like cells. Osteoblasts were cultured in α–minimum essential medium containing 10% fetal bovine serum with or without 100 units/ml of IL-1α for up to 14 days. The gene and protein expression of M-CSF and OPG were estimated by determining mRNA levels using the real-time polymerase chain reaction and protein levels using Western blot analysis. PGE 2 expression was determined using an enzyme-linked immunosorbent assay. The formation of osteoclast-like cells was estimated using tartrate-resistant acid phosphatase (TRAP) staining of osteoclast precursors in culture with conditioned medium from IL-1α-treated osteoblasts and the soluble receptor activator of NF-κB ligand (RANKL). M-CSF and PGE 2 expression in osteoblasts increased markedly in cells cultured with IL-1α, whereas OPG expression decreased. The conditioned medium containing M-CSF and PGE 2 produced by IL-1α-treated osteoblasts and soluble RANKL increased the TRAP staining of osteoclast precursors. These results suggest that IL-1α stimulated the formation of osteoclast-like cells via an increase in M-CSF and PGE 2 production, and a decrease in OPG production by osteoblasts.

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.

IL-1.ALPHA. Stimulates the Formation of Osteoclast-like Cells via RANK-RANKL Signaling System by Osteoblasts and Monocytes

We examined the effect of the inflammatory mediator interleukin-1α (IL-1α) on the expression of macrophage colony-stimulating factor (M-CSF), osteoprotegerin (OPG), and prostaglandin E2 (PGE2) in rat osteoblasts, and the indirect effect of IL-1α on the formation of osteoclast-like cells. The expression of M-CSF and OPG were estimated by determining protein levels using Western blot analysis. PGE2 expression was determined using ELISA. The formation of osteoclast-like cells was estimated using tartrate-resistant acid phosphatase (TRAP) staining of osteoclast precursors in culture with conditioned medium from IL-1α-treated osteoblasts and the soluble receptor activator of NF-κB ligand (RANKL). M-CSF and PGE2 expression in osteoblasts increased markedly in cells cultured with IL-1α, whereas OPG expression decreased. The conditioned medium containing M-CSF and PGE2 produced by IL-1α-treated osteoblasts and soluble RANKL increased the TRAP staining of osteoclast precursors. These results suggest that IL-1α stimulated the formation of osteoclast-like cells via an increase in M-CSF and PGE2 production, and a decrease in OPG production by osteoblasts.

Enamel matrix derivative promotes osteoclast-like cell formation by RANKL production in mouse marrow cultures

Enamel matrix derivative promotes osteoclast-like cell formation by RANKL production in mouse marrow cultures

Ascorbic acid inhibits the formation and function of osteoclasts from RAW264.7 cells induced by receptor activated nuclear factor kappaB ligand in vitro

To observe the influence of ascorbic acid (AA) on osteoclastogenesis induced by receptor activated nuclear factor kappaB ligand (RANKL) in RAW264.7 cells and to study the mechanism of AA in osteoclast differentiation. Mouse mononuclear cells of the line RAW264.7 were cultured. AA and/or RANKL were added. Cellular respiration of RAW264.7 cells was assayed by MTT cell proliferation method. RAW264.7 cells were inoculated and RANKL and AA of different concentrations were added. TRAP staining was used to observe the formation of osteoclasts. RAW264.7 cells were inoculated on OAAS, RANKL and AA were added. The number of bone absorption pits was counted. RT-PCR was used to measure the expression of RANK, matrix metalloproteinase 9, TRAF6, TRAP, integrin alpha v, integrin beta 3, cathepsin K, and carbonic anhydrase II (CA II). AA of the concentration of 500 microg/ml significantly inhibited the proliferation of RAW264.7 cells (0.13 +/- 0.05 vs 1.11 +/- 0.22, P < 0.05). RANKL significantly inhibited the proliferation of RAW264.7 cells as well (1.58 +/- 0.22 vs 1.26 +/- 0.17). AA showed no synergistic action to RANKL. AA alone could not induce the formation of TRAP-positive osteoclasts from RAW264.7 cells and 0 approximately 100 microg/ml AA inhibited the RANKL-induced formation of osteoclast-like multinuclear cells from RAW264.7 cells. AA alone up-regulated the mRNA expression of CA II, cathepsin K, and TRAP by 10, 1.5, and 1.5 times respectively. RANKL plus AA significantly down-regulated the mRNA expression of CAII and RANK by 60% and 20% respectively. No bone absorption pit was seen after addition of AA. The number of pits on OAAS after addition of AA + RANKL was significantly lower than that after addition of RANKL alone (P < 0.05). Western blotting showed that no difference was seen in the protein expression of CA I between the RANKL group and the control group, however, the protein expression of CA II in the low concentration AA + RANKL group was significantly lower than that in the RANKL alone group (P < 0.05). AA directly inhibits RANKL induced osteoclast formation and its function in vitro.

Regucalcin stimulates osteoclast‐like cell formation in mouse marrow cultures

The effect of regucalcin, a regulatory protein in intracellular signaling, on osteoclastic cell formation in mouse bone marrow culture is investigated. The bone marrow cells were cultured for 7 days in an alpha-minimal essential medium containing either vehicle or regucalcin (10(-10)-10(-8)M). Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of regucalcin (10(-10)-10(-8)M) caused a remarkable increase in osteoclast-like multinucleated cells (MNCs). The effect of regucalcin in stimulating osteoclast-like cell formation was significantly inhibited in the presence of calcitonin (CT; 10(-9)M), 17beta-estradiol (10(-9)M), beta-cryptoxanthin (CX; 10(-6)M), or zinc sulfate (10(-4)M), which is an anti-bone resorbing factor. The effect of regucalcin on osteoclast-like cell formation was not significantly blocked in the presence of cycloheximide, an inhibitor of protein synthesis, or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), an inhibitor of transcriptional activity. The effect of parathyroid hormone (10(-7)M), 1,25-dihydroxyvitamin D(3) (10(-7)M), prostaglandin E(2) (10(-5)M), or tumor necrosis factor-alpha (10 ng/ml) in increasing osteoclast-like cell formation was significantly enhanced in the presence of regucalcin (10(-8)M). Moreover, when rat femoral-diaphyseal or -metaphyseal tissues were cultured for 48 h in the presence of regucalcin (10(-10)-10(-8)M), the diaphyseal or metaphyseal calcium content was significantly decreased in the presence of regucalcin (10(-10)-10(-8)M) in vitro. The consumption of glucose and the production of lactic acid in culture medium by the diaphyseal or metaphyseal tissues was significantly raised in the presence of regucalcin (10(-10)-10(-8)M). This study demonstrates that regucalcin directly stimulates osteoclast-like cell formation in mouse marrow culture in vitro, and that the protein stimulates bone resorption in rat femoral tissues in vitro.

Runx-2 is not essential for the vitamin D-regulated expression of RANKL and osteoprotegerin in osteoblastic cells

The differentiation and activity of osteoclasts are positively and negatively controlled by receptor activator of nuclear factor-κB ligand (RANKL), which is expressed on the surface of osteoblasts and stromal cells, and its decoy receptor osteoprotegerin (OPG), which is secreted by osteoblasts and stromal cells, respectively. The expression of the genes for RANKL and OPG is regulated by 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3]. Runt-related gene-2 (Runx-2) is essential for osteoblast differentiation and there are several reports that Runx-2 is involved in osteoclast formation. Therefore, to clarify the role of Runx-2 in osteoclastogenesis, we designed a series of experiments using C2 cells and C6 cells, which are derived from calvariae of runx2-deficient mice. Treatment of C2 cells and C6 cells with 1α,25(OH)2D3 for 2–4 days increased and decreased the levels of expression of the mRNAs for RANKL and OPG, respectively, and the effects were dose-dependent. However, by day 8, the level of RANKL mRNA had fallen and that of OPG mRNA had risen. Furthermore, C6 cells induced the differentiation of mouse spleen cells into tartrate-resistant acid phosphatase-positive (TRAP-positive) multinucleated cells (osteoclast-like cells) in the presence of 10−7M 1α,25(OH)2D3. Such formation of osteoclast-like cells was inhibited by exogenous OPG in a dose-dependent manner. Thus, our findings indicate that Runx-2 is not essential for the expression of RANKL and OPG, and the formation of osteoclast-like cells.

Receptor activator of NF-κB ligand-stimulated osteoclastogenesis in mouse marrow culture is suppressed by zinc in vitro

Zinc has been shown to have an inhibitory effect on osteoclastic bone resorption in vitro. This study was undertaken to determine whether the inhibitory action of zinc on osteoclastogenesis is related to receptor activator of NF-kappaB ligand (RANKL), which plays a pivotal role in differentiation from pre-osteoclasts to osteoclasts. Mouse marrow cells were cultured for 3 days in alpha-minimal essential medium containing lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNFalpha), or RANKL, which stimulates osteoclastogenesis; then zinc sulfate was added to the culture medium containing each osteoclastogenesis-stimulating factor, and the cells were further incubated for 4 days. Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of LPS (10 micro g/ml), TNFalpha (10 ng/ml), or RANKL (10 or 20 ng/ml) with M-CSF (10 or 20 ng/ml) induced a remarkable increase in osteoclast-like multinucleated cells (MNCs). The stimulatory effect of LPS was not significantly altered by the addition of zinc sulfate (10(-6)-10(-4) M). Meanwhile, TNFalpha- or RANKL-induced osteoclast-like cell formation was significantly inhibited in the presence of zinc sulfate (10(-6)-10(-4) M). The effect of zinc sulfate (10(-4) M) in inhibiting RANKL-induced osteoclast-like cell formation was completely abolished in the presence of cycloheximide (10(-7) M), an inhibitor of translation in protein synthesis, or 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB; 10(-6) M), an inhibitor of transcription. These results suggest that the inhibitory action of zinc on osteoclastogenesis is partly due to suppressing signaling pathway which is related to RANKL stimulation in osteoclast development.

(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.

Lipopolysaccharide stimulates expression of osteoprotegerin and receptor activator of NF-kappa B ligand in periodontal ligament fibroblasts through the induction of interleukin-1 beta and tumor necrosis factor-alpha

Our recent work showed that human periodontal ligament fibroblasts (HPLF) secrete bioactive osteoprotegerin (OPG), which inhibits osteoclastic differentiation and activity. However, it is unknown how HPLF regulate bone metabolism in the presence of lipopolysaccharide (LPS), which is a cell component of gram-negative bacteria and a pathogen in inflammatory bone diseases such as periodontitis. The present study examined the effects of Escherichia coli LPS on the gene expression of interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), OPG, and receptor activator of NF-kappa B ligand (RANKL) in HPLF using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. In HPLF cultured with LPS for 48 h, expression of both OPG and RANKL mRNA was up-regulated, whereas for up to 24 h of stimulation, such up-regulation was not observed. However, LPS increased expression of IL-1β and TNF-α mRNA within 6 h of treatment. Moreover, in HPLF cultured with IL-1β or TNF-α, OPG and RANKL expression was induced within 12 h of culture. The administration of neutralizing antibodies against human IL-1β or TNF-α to LPS-treated cultures of HPLF inhibited the induction of OPG and RANKL expression. These suggest that LPS stimulates both OPG and RANKL expression in HPLF by up-regulating IL-1β and TNF-α. In addition, administration of conditioned medium (CM) from HPLF (HPLF-CM) stimulated with LPS for 48 h to mouse bone marrow culture failed to induce osteoclast-like cell (OCL) formation. When mouse spleen cells were cocultured with HPLF in the presence of LPS, OCL formation was completely blocked. Taken together, our results indicate that human periodontal ligament cells stimulated with LPS inhibit osteoclastogenesis by producing more effective OPG than RANKL via the induction of IL-1β and TNF-α.