Production of IL-7 is increased in ovariectomized mice,but not RANKL mRNA expression by osteoblasts/stromal cells in bone,and IL-7 enhances generation of osteoclast precursors in vitro

Osteoclastogenic cytokines produced by T and B lineage cells and interleukin (IL)-7-induced expansion of the pool size of osteoclast precursors have been suggested to play an important role in acceleration of osteoclastogenesis induced by estrogen deficiency. However, the contribution of increased RANKL produced by osteoblasts/stromal cells to increase osteoclastogenesis in a mouse model of estrogen-deficient osteoporosis and in vitro effects of IL-7 on osteoclast precursor generation remain controversial. Thus, we investigated the effect of ovariectomy (OVX) of mice on production of RANKL, osteoprotegerin (OPG), and IL-7 in bone and the effect of IL-7 on osteoclast precursor generation in vitro. OVX did not significantly stimulate mRNA expressions of RANKL and OPG in whole femurs. Because the epiphysis, but not the femoral shaft (diaphysis) or bone marrow, is the main site of osteoclastogenesis, it is important to specifically analyze mRNA expression by osteoblasts/stromal cells at these parts of the femur. Therefore, we isolated RNA from bone marrow cell-free epiphysis, diaphysis, and flushed-out bone marrow and examined mRNA expression. The results showed no significant changes of RANKL and OPG mRNA expression in any part of the femur. In addition, OVX did not significantly affect RANKL and OPG mRNA expression by the adherent stromal cells isolated from flushed-out bone marrow cells but did stimulate RANKL mRNA expression by B220⁺ cells in the nonadherent cell fraction. On the other hand, OVX increased IL-7 mRNA expression in the femur as well as IL-7 concentrations in bone fluid. In cultures of unfractionated bone cells isolated by vigorous agitation of minced whole long bones to release the cells tightly attached to the bone surfaces, but not in cocultures of clonal osteoblasts/stromal cells and flushed-out bone marrow cells, IL-7 stimulated generations of osteoclasts as well as osteoclast precursors. These data suggest that increased RANKL production by osteoblasts/stromal cells is unlikely to play a central role in acceleration of osteoclastogenesis in estrogen deficiency of mice and that IL-7 stimulates osteoclast precursor generation, presumably through an action of IL-7 on the cells attached to bone rather than on cells contained in the bone marrow cell population.     

Prostaglandin E2 induces expression of receptor activator of nuclear factor-kappa B ligand/osteoprotegrin ligand on pre-B cells: implications for accelerated osteoclastogenesis in estrogen deficiency.

Estrogen deficiency causes bone loss as a result of accelerated osteoclastic bone resorption. It also has been reported that estrogen deficiency is associated with an increase in the number of pre-B cells in mouse bone marrow. The present study was undertaken to clarify the role of altered B lymphopoiesis and of the receptor activator of nuclear factor-kappa B ligand (RANKL), a key molecule in osteoclastogenesis, in the bone loss associated with estrogen deficiency. In the presence of prostaglandin E2 (PGE2), the activity to form tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells was significantly greater in bone marrow cells derived from ovariectomized (OVX) mice than in those from sham-operated mice. Northern blot analysis revealed that PGE2 increased the amount of RANKL messenger RNA (mRNA) in bone marrow cells, not only adherent stromal cells but nonadherent hematopoietic cells; among the latter, RANKL mRNA was more abundant in OVX mice than in shamoperated mice and was localized predominantly in B220+ cells. Flow cytometry revealed that most B220+ cells in bone marrow were RANKL positive and that the percentage of RANKL-positive, B220low cells was higher in bone marrow from OVX mice than in that from sham-operated mice. The increase in the expression of RANKL and the percentage of these cells in OVX mice was abolished by the administration of indomethacin in vivo. PGE2 also markedly increased both the level of RANKL mRNA and cell surface expression of RANKL protein in the mouse pre-B cell line 70Z/3. Finally, osteoclastogenic response to PGE2 was reduced markedly by prior depletion of B220+ cells, and it was restored by adding back B220+ cells. Taken together with stimulated cyclo-oxygenase (COX)-2 activity by tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1) in estrogen deficiency, these results suggest that an increase in the number of B220+ cells in bone marrow may play an important role in accelerated bone resorption in estrogen deficiency because B220+ cells exhibit RANKL on the cell surface in the presence of PGE2, thereby leading to accelerated osteoclastogenesis.     

Mice lacking the integrin beta5 subunit have accelerated osteoclast maturation and increased activity in the estrogen-deficient state.

Integrin alphavbeta5 is expressed on osteoclast precursors and is capable of recognizing the same amino acid motif as alphavbeta3. Three-month-old beta5(-/-) female OVX mice had increased osteoclastogenesis ex vivo, and microCT assessment of trabecular bone volume was 53% lower than WT-OVX animals. These preliminary data suggest alphavbeta5 integrin's presence on osteoclast precursors may inhibit of osteoclast formation. INTRODUCTION: Osteoclasts are unique resorptive skeletal cells, capable of degrading bone on contact to the juxtaposed matrix. Integrin alphavbeta5 is expressed on osteoclast precursors, structurally similar to alphavbeta3, and capable of recognizing the same amino acid motif. Given the structural relationship and reciprocal regulation of alphavbeta3 and alphavbeta5, the purpose of this study was to evaluate how alphavbeta5 might contribute to osteoclast maturation and activity. MATERIALS AND METHODS: Three-month-old wildtype (WT) and beta5(-/-) female mice had ovariectomy (OVX) or sham operations. The osteoclastogenic capacity of marrow-derived precursors, the kinetic, the circulating, and structural parameters of bone remodeling, was determined after 6 weeks of paired feeding. RESULTS AND CONCLUSIONS: OVX increased osteoclastogenesis ex vivo and in vivo. Osteoclast formation and prolonged pre-osteoclast survival were substantially enhanced in cultures containing beta5(-/-) cells whether obtained from sham-operated or OVX mice. Expression of cathepsin K, beta3 integrin subunit, and calcitonin receptor were accelerated in cultured beta5(-/-)osteoclasts. beta5(-/-) osteoclasts from OVX animals showed a 3-fold enhancement of net resorptive activity, with quantitative muCT showing trabecular bone volume loss after OVX 53% greater in beta5(-/-) OVX compared with similarly treated WT OVX mice (p < 0.05). alpha5beta3 seems to be an inhibitor of osteoclast formation, in contrast to alphavbeta3. In addition, loss of alphavbeta5 seems to accelerate osteoclast formation in the OVX model. Further examination of alphavbeta5 signaling pathways may enhance our understanding of the activation of bone resorption.     

A novel molecular mechanism modulating osteoclast differentiation and function.

Osteoclasts, the multinucleated giant cells that resorb bone, develop from hematopoietic cells of the monocyte/ macrophage lineage. Osteoblasts, as well as bone marrow stromal cells, support osteoclast development through a mechanism of cell-to-cell interaction with osteoclast progenitors. We recently purified and molecularly cloned osteoclastogenesis inhibitory factor (OCIF), which was identical to osteoprotegerin (OPG). OPG/OCIF, a secreted member of the tumor necrosis factor (TNF) receptor family, inhibited differentiation and activation of osteoclasts. A single class of high-affinity binding sites for OPG/OCIF appeared on a mouse bone marrow stromal cell line, ST2, in response to 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] and dexamethasone (Dex). When the binding sites were occupied by OPG/OCIF, ST2 cells failed to support the osteoclast formation from spleen cells. To identify an OPG/OCIF ligand, we screened a cDNA expression library of ST2 cells treated with 1,25(OH)2D3 and Dex using OPG/OCIF as a probe. The cloned molecule was found to be a member of the membrane-associated TNF ligand family, and it induced osteoclast formation from mouse and human osteoclast progenitors in the presence of macrophage colony-stimulating factor (M-CSF) in vitro. Expression of its gene in osteoblasts/stromal cells was up-regulated by osteotropic factors, such as 1,25(OH)2D3, prostaglandin E2 (P(GE2), parathyroid hormone (PTH), and interleukin (IL)-11. A polyclonal antibody against this protein, as well as OPG/OCIF, negated not only the osteoclastogenesis induced by the protein, but also bone resorption elicited by various osteotropic factors in a fetal mouse long bone culture system. These findings led us to conclude that the protein is osteoclast differentiation factor (ODF), a long sought-after ligand that mediates an essential signal to osteoclast progenitors for their differentiation into active osteoclasts. Recent analyses of ODF receptor demonstrated that RANK, a member of the TNF receptor family, is the signaling receptor for ODF in osteoclastogenesis, and that OPG/OCIF acts as a decoy receptor for ODF to compete against RANK. The discovery of ODF, OPG/OCIF, and RANK opens a new era in the investigation of the regulation of osteoclast differentiation and function.     

Fibroblastic stromal cells express receptor activator of NF-kappa B ligand and support osteoclast differentiation.

Osteoclast formation in bone is supported by osteoblasts expressing receptor activator of NF-kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) expression. Numerous osteotropic factors regulate expression levels of RANKL and the RANKL decoy receptor osteoprotegerin (OPG) in osteoblasts, thereby affecting osteoclast differentiation. However, not only in RANKL widely expressed in soft tissues, but osteoclasts have been noted in extraskeletal lesions. We found that cultured skin fibroblastic cells express RANKL, M-CSF, and OPG messenger (mRNA). Stimulation by 1 alpha,25 dihydroxyvitamin D3 [1,25(OH)2D3] plus dexamethasone (Dex) augmented RANKL and diminished OPG mRNA expression in fibroblastic cells and caused the formation of numerous osteoclasts in cocultures of skin fibroblastic cells with hemopoietic cells or monocytes. The osteoclasts thus formed expressed tartrate-resistant acid phosphatase (TRAP) and calcitonin (CT) receptors and formed resorption pits in cortical bone. Osteoclast formation also was stimulated (in the presence of Dex) by prostaglandin E2 (PGE2), interleukin-11 (IL-11), IL-1, tumor necrosis factor-alpha (TNF-alpha), and parathyroid hormone-related protein (PTHrP), factors which also stimulate osteoclast formation supported by osteoblasts. In addition, granulocyte-macrophage-CSF (GM-CSF), transforming growth factor-beta (TGF-beta), and OPG inhibited osteoclast formation in skin fibroblastic cell-hemopoietic cell cocultures; CT reduced only osteoclast nuclearity. Fibroblastic stromal cells from other tissues (lung, respiratory diaphragm, spleen, and tumor) also supported osteoclast formation. Thus, RANKL-positive fibroblastic cells in extraskeletal tissues can support osteoclastogenesis if osteolytic factors and osteoclast precursors are present. Such mesenchymally derived cells may play a role in pathological osteolysis and may be involved in osteoclast formation in extraskeletal tissues.     

The generation of highly enriched osteoclast-lineage cell populations.

Osteoclasts form when hematopoietic cells are stimulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL) or tumor necrosis factor-alpha (TNFalpha). Osteoclast precursors derive from M-CSF-dependent proliferating hematopoietic cells but cannot yet be purified from mixed populations. M-CSF stimulation of bone marrow cells results in large numbers of nonadherent, proliferating macrophage precursors. These rapidly form adherent bone marrow macrophages (BMM). BMM and their precursors can be isolated free from mesenchymal and lymphocytic cells. BMM precursors derived from CBA-strain mouse bone marrow, when cocultured with ST2 cells (which express RANKL and M-CSF), formed numerous mononuclear osteoclasts, which resorbed bone and expressed tartrate-resistant acid phosphatase (TRAP) and calcitonin receptors (CTR). Addition of approximately 10 BMM precursors to ST2 cultures resulted in over 80% of these cocultures forming functional osteoclasts, suggesting that they are a highly enriched source of osteoclast progenitors. Supporting this, recombinant RANKL/M-CSF-stimulated BMM precursors formed populations in which all cells expressed TRAP. While only a small proportion of these cells (8.6%) expressed CTR, with transforming growth factor-beta (TGFbeta) present RANKL/M-CSF-stimulated BMM precursors formed almost pure (98.4%) CTR-positive osteoclasts after 7 days. This suggests that TGFbeta stimulated the maturation rate of these cells. Passaged or viably frozen BMM precursors gave rise to BMM that also all formed osteoclasts lineage cells after RANKL/M-CSF stimulation. These data suggest that BMM precursors derived from CBA mice are an expanded pool of osteoclast progenitors. These can be employed to generate osteoclast populations of high purity and in large numbers when stimulated by TGFbeta, which greatly augments the osteoclastogenic effects of RANKL.     

Indapamide, a thiazide-like diuretic, decreases bone resorption in vitro.

We recently showed that indapamide (IDP), a thiazide-related diuretic, increases bone mass and decreases bone resorption in spontaneously hypertensive rats supplemented with sodium. In the present study, we evaluated the in vitro effects of this diuretic on bone cells, as well as those of hydrochlorothiazide (HCTZ), the reference thiazide, and acetazolamide (AZ), a carbonic anhydrase (CA) inhibitor. We showed that 10(-4) M IDP and 10(-4) M AZ, as well as 10(-5) M pamidronate (APD), decreased bone resorption in organ cultures and in cocultures of osteoblast-like cells and bone marrow cells in the presence of 10(-8) M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We investigated the mechanism of this antiresorptive effect of IDP; IDP decreased osteoclast differentiation as the number of osteoclasts developing in coculture of marrow and osteoblast-like cells was decreased markedly. We then investigated whether IDP affected osteoblast-like cells because these cells are involved in the osteoclast differentiation. Indeed, IDP increased osteoblast-like cell proliferation and alkaline phosphatase (ALP) expression. Nevertheless, it did not modify the colony-stimulating factor 1 (CSF-1) production by these cells. In addition, osteoblast-like cells expressed the Na+/Cl- cotransporter that is necessary for the renal action of thiazide diuretics, but IDP inhibited bone resorption in mice lacking this cotransporter, so the inhibition of bone resorption and osteoclast differentiation did not involve this pathway. Thus, we hypothesized that IDP may act directly on cells of the osteoclast lineage. We observed that resorption pits produced by spleen cells cultured in the presence of soluble osteoclast differentiation factor (sODF) and CSF-1 were decreased by 10(-4) M IDP as well as 10(-5) M APD. In conclusion, in vitro IDP increased osteoblast proliferation and decreased bone resorption at least in part by decreasing osteoclast differentiation via a direct effect on hematopoietic precursors.     

Impaired bone resorption in cathepsin K-deficient mice is partially compensated for by enhanced osteoclastogenesis and increased expression of other proteases via an increased RANKL/OPG ratio

Previous reports indicate that mice deficient for cathepsin K (Ctsk), a key protease in osteoclastic bone resorption, develop osteopetrosis due to their inability to properly degrade organic bone matrix. Some features of the phenotype of Ctsk knockout mice, however, suggest the presence of mechanisms by which Ctsk-deficient mice compensate for the lack of cathepsin K. To study these mechanisms in detail, we generated Ctsk-deficient (Ctsk-/-) mice and analyzed them at the age of 2, 7, and 12 months using peripheral quantitative computed tomography, histomorphometry, resorption marker measurements, osteoclast and osteoblast differentiation cultures, and gene expression analyses. The present study verified the previously published osteopetrotic features of Ctsk-deficient mice. However, these changes did not exacerbate during aging indicating the absence of Ctsk to have its most severe effects during the rapid growth period. Resorption markers ICTP and CTX were decreased in the media of Ctsk-/- osteoclasts cultured on bone slices indicating impaired bone resorption. Ctsk-/- mice exhibited several mechanisms attempting to compensate for Ctsk deficiency. The number of osteoclasts in trabecular bone was significantly increased in Ctsk-/- mice compared to controls, as was the number of osteoclast precursors in bone marrow. The mRNA levels for receptor activator of nuclear factor (kappa)B ligand (RANKL) in Ctsk-/- bones were increased resulting in increased RANKL/OPG ratio favoring osteoclastogenesis. In addition, expression of mRNAs of osteoclastic enzymes (MMP-9, TRACP) and for osteoblastic proteases (MMP-13, MMP-14) were increased in Ctsk-/- mice compared to controls. Impaired osteoclastic bone resorption in Ctsk-/- mice results in activation of osteoblastic cells to produce increased amounts of other proteolytic enzymes and RANKL in vivo. We suggest that increased RANKL expression mediates enhanced osteoclastogenesis and increased protease expression by osteoclasts. These observations underline the important role of osteoblastic cells in regulation of osteoclast activity and bone turnover.     

TNFα inhibits the development of osteoclasts through osteoblast-derived GM-CSF

Inflammatory cytokines such as tumor necrosis factor-alpha (TNFα) are potent stimulators of osteoclast formation and bone resorption and are frequently associated with pathologic bone metabolism. The cytokine exerts specific effects on its target cells and constitutes a part of the cellular microenvironment. Previously, TNFα was demonstrated to inhibit the development of osteoclasts in vitro via an osteoblast-mediated pathway. In the present study, the molecular mechanisms of the inhibition of osteoclastogenesis were investigated in co-cultures of osteoblasts and bone marrow cells (BMC) and in cultures of macrophage-colony stimulating factor (M-CSF) dependent, non-adherent osteoclast progenitor cells (OPC) grown with M-CSF and receptor activator of NF-κB ligand (RANKL). Granulocyte-macrophage colony stimulating factor (GM-CSF), a known inhibitor of osteoclastogenesis was found to be induced in osteoblasts treated with TNFα and the secreted protein accumulated in the supernatant. Dexamethasone (Dex), an anti-inflammatory steroid, caused a decrease in GM-CSF expression, leading to partial recovery of osteoclast formation. Flow cytometry analysis revealed that in cultures of OPC, supplemented with 10% conditioned medium (CM) from osteoblasts treated with TNFα/1,25(OH)(2)D(3), expression of RANK and CD11c was suppressed. The decrease in RANK expression may be explained by the finding, that GM-CSF and the CM from wt osteoblasts were found to suppress the expression of c-Fos, Fra-1, and Nfatc-1. The failure of OPC to develop into CD11c(+) dendritic cells suggests that cell development is not deviated to an alternative differentiation pathway, but rather, that the monocytes are maintained in an undifferentiated, F4/80(+), state. The data further implies possible interactions among inflammatory cytokines. GM-CSF induced by TNFα acts on early hematopoietic precursors, inhibiting osteoclastogenesis while acting as the growth factor for M-CSF independent inflammatory macrophages. These in turn may condition a microenvironment enhancing osteoclast differentiation and bone resorption upon migration of the OPC from circulation to the bone/bone marrow compartment.     

Comparative effects of estrogen and raloxifene on B lymphopoiesis and bone loss induced by sex steroid deficiency in mice.

Estrogen deficiency caused by ovariectomy (OVX) results in a marked bone loss because of stimulated bone resorption. We have reported that OVX selectively stimulates B lymphopoiesis in mouse bone marrow, which is somehow related to bone resorption. Estrogen prevents both the increased B lymphopoiesis and the bone resorption caused by estrogen deficiency. Raloxifene also has a potent estrogenic activity for bone with minimal estrogenic activity for the uterus. To examine the effects of raloxifene on B lymphopoiesis and bone resorption, OVX mice were given either estrogen or raloxifene subcutaneously for 2-4 weeks using a miniosmotic pump. Reduced uterine weight in OVX mice was restored completely by 17beta-estradiol (E2). Some 300-fold higher doses of raloxifene increased uterine weight of OVX mice, but only slightly. The number of B220- positive pre-B cells was increased markedly in bone marrow after OVX. The increased B lymphopoiesis was prevented not only by E2 but by raloxifene. In OVX mice, the trabecular bone volume (BV) of the femoral distal metaphysis was reduced markedly, when measured by microcomputed tomography (microCT) scanning and dual-energy X-ray absorptiometry. Both E2 and raloxifene similarly restored it. Like estrogen deficiency, androgen deficiency induced by orchidectomy (ORX) also resulted in a marked bone loss and increased B lymphopoiesis. Both E2 and raloxifene prevented the changes in ORX mice. These results indicate that both estrogen deficiency and androgen deficiency similarly stimulate B lymphopoiesis in mouse bone marrow, which accompany bone loss. Raloxifene exhibits estrogenic actions in bone and bone marrow to prevent bone loss and regulate B lymphopoiesis without inducing estrogenic action in the uterus.     

Inhibitory and stimulatory effects of prostaglandins on osteoclast differentiation

The effect of prostaglandins (PGs) on osteoclast differentiation, an important point of control for bone resorption, is poorly understood. After an initial differentiation phase that lasts at least 4 days, murine monocytes, cocultured with UMR106 osteoblastic cells (in the presence of 1,25-dihydroxyvitamin D₃) give rise to tartrate-resistant acid phosphatase (TRAP) positive osteoclast-like cells that are capable of lacunar bone resorption. PGE₂ strongly inhibits TRAP expression and bone resorption in these cocultures. To examine further the cellular mechanisms associated with this inhibitory effect, we added PGE₂ to monocyte/UMR106 cocultures at specific times before, during, and after this initial 4-day differentiation period. To determine whether this PGE₂ inhibition was dependent on the type of stromal cell supporting osteoclast differentiation, we also added PGE₂ to cocultures of monocytes with ST2 preadipocytic cells. Inhibition of bone resorption was greatly reduced when the addition of PGE₂ to monocyte/UMR106 cocultures was delayed until the fourth day of incubation; when delayed until the seventh day, inhibition did not occur. PGE₂ inhibition of bone resorption was concentration-dependent and at 10⁻⁶ M was also mediated by PGE₁ and PGF_2α. In contrast to its effects on monocyte/UMR106 cocultures, PGE₂ stimulated bone resorption in monocyte/ST2 cocultures. Both ST2 cells and UMR106 cells were shown to express functional receptors for PGE₂. These results show that PGs strongly influence the differentiation of osteoclast precursors and that this effect is dependent not only on the type and dose of PG administered, but also on the nature of the bone-derived stromal cell supporting this process. Received: 12 October 1995 / Accepted: 1 April 1996     

Osteoclastogenesis is Influenced by Modulation of Gap Junctional Communication with Antiarrhythmic Peptides

Osteoclasts are formed by the fusion of mononuclear precursor cells of the monocyte–macrophage lineage. Among several putative mechanisms, gap-junctional intercellular communication (GJC) has been proposed to have a role in osteoclast fusion and bone resorption. We examined the role of GJC in osteoclastogenesis and in vitro bone resorption with mouse bone marrow hematopoietic stem cells and RAW 264.7 cells. Blocking of gap junctions with 18-α-glycyrrhetinic acid (18GA) led to inhibition of osteoclastogenesis and in vitro bone resorption. Similarly, the GJC inhibitor GAP27 inhibited osteoclast formation. GJC modulation with the antiarrhythmic peptides (AAPs) led to increased amounts of multinuclear RAW 264.7 osteoclasts as well as increased number of nuclei per multinuclear cell. In the culture of bone marrow hematopoietic stem cells in the presence of bone marrow stromal cells AAP reduced the number of osteoclasts, and coculture of MC3T3-E1 preosteoblasts with RAW 264.7 macrophages prevented the action of AAPs to promote osteoclastogenesis. The present data indicate that AAPs modulate the fusion of the pure culture of cells of the monocyte–macrophage lineage. However, the fusion is influenced by GJC in cells of the osteoblast lineage.     

The effect of particle phagocytosis and metallic wear particles on osteoclast formation and bone resorption in vitro

Osteoclasts are multinucleated bone-resorbing cells that are formed from precursors that circulate in the monocyte fraction. This study has determined the effect of phagocytosis of metal particles on osteoclast formation and bone resorption in vitro. Human peripheral blood monocytes were cocultured for 21 days with osteoblast-like UMR 106 cells, in the presence of 1,25-dihydroxyvitamin D3, dexamethasone, and human macophage colony-stimulating factor. Cobalt-chrome alloy (CoCr), stainless steel (316L-SS), titanium alloy (TiAlV), and commercially pure titanium (cpTi) particles (size range, 0.5-3.0 microm) and 1.0-microm latex particles were added to the cocultures as a single dose at the beginning of each experiment. All 5 types of particles were readily phagocytosed by the monocytes. After 4 days' exposure to high concentrations of all the metal particles, some cell death was found in the cocultures. After 14 days, a reduction in the number of CD14+ cells was seen in cocultures exposed to high concentrations of metal particles, particularly CoCr and 316L-SS particles. Phagocytosis of latex particles by osteoclast precursors did not affect the ability of these cells to undergo osteoclast differentiation. In contrast, exposure to metal wear particle preparations caused a dose-dependent reduction in the number of vitronectin receptor-positive osteoclastic cells formed and a dose-dependent reduction in the bone resorption produced by these cells. This decrease in resorption was greater after exposure to CoCr and 316L-SS particles compared with TiAlV and cpTi particles. This in vitro cell culture system may provide a useful model to compare the effect of different prosthetic materials on human osteoclast formation and bone resorption.     

A DNA microarray analysis of chemokine and receptor genes in the rat dental follicle--role of secreted frizzled-related protein-1 in osteoclastogenesis

The dental follicle, a loose connective tissue sac that surrounds the unerupted tooth, appears to regulate the osteoclastogenesis needed for eruption; i.e., bone resorption to form an eruption pathway. Thus, DNA microarray studies were conducted to determine which chemokines and their receptors were expressed chronologically in the dental follicle, chemokines that might attract osteoclast precursors. In the rat first mandibular molar, a major burst of osteoclastogenesis occurs at day 3 with a minor burst at day 10. The results of the microarray confirmed our previous studies showing the gene expression of molecules such as CSF-1 and MCP-1 in the dental follicle cells. Other new genes also were detected, including secreted frizzled-related protein-1 (SFRP-1), which was found to be downregulated at days 3 and 9. Using rat bone marrow cultures to conduct in vitro osteoclastogenic assays, it was demonstrated that SFRP-1 inhibited osteoclast formation in a concentration-dependent fashion. However, with increasing concentrations of SFRP-1, the number of TRAP-positive mononuclear cells increased suggesting that SFRP-1 inhibits osteoclast formation by inhibiting the fusion of mononuclear cells (osteoclast precursors). Co-culturing bone marrow mononuclear cells and dental follicle cells demonstrated that the dental follicle cells were secreting a product(s) that inhibited osteoclastogenesis, as measured by counting of TRAP-positive osteoclasts. Adding an antibody either to SFRP-1 or OPG partially restored osteoclastogenesis. Adding both anti-SFRP-1 and anti-OPG fully negated the inhibitory effect of the follicle cells upon osteoclastogenesis. These results strongly suggest that SFRP-1 and OPG, both secreted by the dental follicle cells, use different pathways to exert their inhibitory effect on osteoclastogenesis. Based on these in vitro studies of osteoclastogenesis, it is likely that the downregulation of SFRP-1 gene expression in the dental follicle at days 3 and 9 is a contributory factor in allowing the major and minor bursts of osteoclastogenesis to occur. Thus, inhibition of SFRP-1 gene expression in combination with inhibition of OPG gene expression likely are critical events in enabling alveolar bone resorption to occur such that teeth will erupt.     

Bisphosphonates suppress bone resorption by a direct effect on early osteoclast precursors without affecting the osteoclastogenic capacity of osteogenic cells: the role of protein geranylgeranylation in the action of nitrogen-containing bisphosphonates on osteoclast precursors.

Nitrogen-containing bisphosphonates (NBps) are taken up by osteoclasts and inhibit farnesyl pyrophosphate synthase, an enzyme of the mevalonate pathway. There is evidence, however, that cells other than mature osteoclasts, like osteoclast precursors and osteoblasts, are also involved in the action of Bps on bone resorption in vitro. To examine this issue further, we developed a new in vitro model, which allows the study of the effects of additives on early osteoclast precursors. In this model, osteogenic cells are essential for osteoclastogenesis. The model consists of 15-day-old fetal mouse metatarsals. At time of explantation, these bone rudiments do not yet contain a mineralized matrix or osteoclasts; only early osteoclast precursors are present in the perichondrium. During culture and after the addition of Nabeta-glycerolphosphate, the bones form a mineralized matrix that is consequently resorbed by osteoclasts that develop from their precursors. Short treatment of these explants with Bps, before the formation of a mineralized matrix, resulted in a subsequent dose-dependent inhibition of bone resorption. The relative potencies of eight Bps to suppress resorption were comparable with those observed after the addition of Bps after the formation of a mineralized matrix, the natural target of Bps. In addition, the effects of the NBp olpadronate, but not of clodronate, on osteoclastic resorption, could be partly reversed by geranylgeraniol. Results indicate that Bps can suppress osteoclastic resorption in vitro by a direct action on very early osteoclast precursors at the bone surface, and not by affecting the osteoclastogenic capacity of osteogenic cells. Moreover, the mechanism of action of the NBp olpadronate, but not clodronate, on early tartrate-resistant acid phosphatase-negative osteoclast precursors involves inhibition of protein geranylgeranylation, indicating a molecular mechanism similar to that established for mature osteoclasts.     

Tumor necrosis factor-alpha: alternative role as an inhibitor of osteoclast formation in vitro

TNFalpha is known to stimulate the development and activity of osteoclasts and of bone resorption. The cytokine was found to mediate bone loss in conjunction with inflammatory diseases such as rheumatoid arthritis or chronic aseptic inflammation induced by wear particles from implants and was suggested to be a prerequisite for the loss of bone mass under estrogen deficiency. In the present study, the regulation of osteoclastogenesis by TNFalpha was investigated in co-cultures of osteoblasts and bone marrow or spleen cells and in cultures of bone marrow and spleen cells grown with CSF-1 and RANKL. Low concentrations of TNFalpha (1 ng/ml) caused a >90% decrease in the number of osteoclasts in co-cultures, but did not affect the development of osteoclasts from bone marrow cells. In cultures with p55TNFR(-/-) osteoblasts and wt BMC, the inhibitory effect was abrogated and TNFalpha induced an increase in the number of osteoclasts in a dose-dependent manner. Osteoblasts were found to release the inhibitory factor(s) into the culture supernatant after simultaneous treatment with 1,25(OH)(2)D(3) and TNFalpha, this activity, but not its release, being resistant to treatment with anti-TNFalpha antibodies. Dexamethasone blocked the secretion of the TNFalpha-dependent inhibitor by osteoblasts, while stimulating the development of osteoclasts. The data suggest that the effects of TNFalpha on the differentiation of osteoclast lineage cells and on bone metabolism may be more complex than hitherto assumed and that these effects may play a role in vivo during therapies for inflammatory diseases.     

Isolation of human osteoclasts formed in vitro: hormonal effects on the bone-resorbing activity of human osteoclasts

Osteoclasts are multinucleated cells that carry out bone resorption. Analysis of the direct effect of hormones on the bone-resorbing activity of human osteoclasts has been limited by difficulties in isolating these cells from the human skeleton. In this study, human osteoclasts formed from cultures of peripheral blood mononuclear precursors (PBMCs) on a Type-I collagen gel were isolated by collagenase treatment for investigating their resorptive activity. PBMCs were cultured in the presence of M-CSF, soluble RANKL, dexamethasone, and 1,25(OH)2D3. The isolated multinucleated cells expressed the osteoclast markers, TRAP, VNR, cathepsin K, calcitonin receptors and were capable of extensive lacunar resorption. Calcitonin inhibited the motility and resorptive activity of osteoclasts. RANKL significantly stimulated osteoclast resorption, but 1,25(OH)2D3, PTH, and OPG did not. These findings indicate that calcitonin and RANKL act directly on human osteoclasts to inhibit and stimulate osteoclast bone-resorbing activity, respectively, and that PTH, 1,25(OH)2D3, and OPG are more likely to influence osteoclast activity indirectly. This technique of human osteoclast isolation should permit the effects of cellular and hormonal/humoral factors on the bone-resorbing activity of mature human osteoclasts to be assessed independently of any effect such factors have on osteoclast formation. It should also make it possible to examine directly the resorptive activity and other characteristics of osteoclasts in specific bone disorders such as Paget's disease.     

The increased in vitro osteoclastogenesis in patients with rheumatoid arthritis is due to increased percentage of precursors and decreased apoptosis - the In Vitro Osteoclast Differentiation in Arthritis (IODA) study

Increases in local and systemic bone resorption are hallmarks of rheumatoid arthritis (RA). Osteoclasts are implicated in these processes and their enhanced differentiation may contribute to bone destruction. We observed that in vitro osteoclastogenesis varies among healthy individuals and hypothesized that increased osteoclastogenesis could be a marker for the presence of RA. Our objective in the present study was to determine if in vitro osteoclastogenesis from peripheral blood mononuclear cells (PBMCs) was different in patients with RA compared to healthy controls and osteoarthritis (OA) patients. Expression of CD14 in PBMCs was quantified and PBMCs were incubated for 21 days in the presence of the osteoclastogenic cytokines M-CSF and RANKL. Differentiation on cortical bone slices permitted the analysis of bone resorption while apoptotic potential was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. In vitro osteoclastogenesis was higher in PBMCs from RA patients compared to controls, and a similar increase was observed in the percentage of osteoclast precursors in RA patients. Osteoclasts from RA patients showed lower apoptotic rates than osteoclasts from healthy controls. No difference was observed in bone resorption activity between RA patients and controls. Interestingly, the difference in osteoclast number and apoptosis rate allowed the implementation of an algorithm capable of distinguishing patients with RA from controls. In conclusion, our study shows that osteoclast differentiation from PBMCs is enhanced in patients with RA, and this difference can be explained by both a higher percentage of osteoclast precursors in the blood and by the reduced apoptotic potential of mature osteoclasts.     

1,25-dihydroxyvitamin D3 inhibits directly human osteoclastogenesis by down-regulation of the c-Fms and RANK expression

Objective1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) is a key molecule to maintain calcium homeostasis and bone metabolism. It was recently reported that 1,25(OH)₂D₃ directly inhibited osteoclast differentiation in mouse bone marrow cells and human bone marrow-derived colony-forming unit granulocyte macrophage (CFU-GM) cells. However, the direct effects of 1,25(OH)₂D₃ and its affecting mechanisms on the osteoclast differentiation of human osteoclast precursors remain largely unknown. In this study, we examined the direct effects of 1,25(OH)₂D₃ on the osteoclastogenesis of human peripheral blood (PB) osteoclast precursors.MethodsIn vitro osteoclastogenesis assays were performed using osteoclast precursors from normal PB. The gene expressions were analyzed using real-time PCR. The cell surface proteins, including c-Fms and RANK, were measured by flow cytometry.Results1,25(OH)₂D₃ strongly inhibited osteoclast differentiation and it suppressed the expression of RANK in the human PB osteoclast precursors. One mechanism of RANK inhibition by 1,25(OH)₂D₃ is down-regulation of the M-CSF receptor c-Fms, which is required for the expression of RANK. In contrast to the previous reports on mouse osteoclast precursors, 1,25(OH)₂D₃ did not affect the expression of c-Fos. Parallel to the inhibition of osteoclastogenesis, 1,25(OH)₂D₃ increased the expression and phosphorylation of CCAAT enhancer-binding protein β (C/EBPβ), which is a recently discovered inhibitor of osteoclastogenesis.ConclusionsOur results show that 1,25(OH)₂D₃ inhibits human osteoclastogenesis by decreasing the RANK+ osteoclast precursors, and we suggest that 1,25(OH)₂D₃ may be a powerful therapeutic agent for treating inflammation-induced bone disease that shows excessive osteoclast activation.     

MiR‐503 Regulates Osteoclastogenesis via Targeting RANK

MicroRNAs (miRNAs) play important roles in osteoclastogenesis and bone resorption. However, no study has investigated the role of miRNA in postmenopausal osteoporosis. Here, we report that miR‐503 was markedly reduced in circulating progenitors of osteoclasts–CD14⁺ peripheral blood mononuclear cells (PBMCs) from postmenopausal osteoporosis patients compared with those from postmenopausal healthy women. Overexpression of miR‐503 in CD14⁺ PBMCs inhibited receptor activator of nuclear factor‐κB ligand (RANKL)‐induced osteoclastogenesis. Conversely, silencing of miR‐503 in CD14⁺ PBMCs promoted osteoclastogenesis. RANK, which is activated by the binding of RANKL and inducing osteoclast differentiation, was confirmed to be a target of miR‐503. In vivo, silencing of miR‐503 using a specific antagomir in ovariectomy (OVX) mice increased RANK protein expression, promoted bone resorption, and decreased bone mass, whereas overexpression of miR‐503 with agomir inhibited bone resorption and prevented bone loss in OVX mice. Thus, our study revealed that miR‐503 plays an important role in the pathogenesis of postmenopausal osteoporosis and contributes to a new therapeutic way for osteoporosis. © 2014 American Society for Bone and Mineral Research.