LPS administration increases CD11b<Superscript>+</Superscript> c-Fms<Superscript>+</Superscript> CD14<Superscript>+</Superscript> cell population that possesses osteoclast differentiation potential in mice

Osteoclasts are multinucleated giant cells that originate from a monocyte/macrophage lineage, and are involved in the inflammatory bone destruction accompanied by periodontitis. Recent studies have shown that osteoclast precursors reside not only in the bone marrow, but also in the peripheral blood and spleen, though the precise characteristics of each precursor have not been analyzed. We hypothesized that the number of osteoclast precursors in those tissues may increase under pathological conditions and contribute to osteoclast formation in vivo in a mouse model. To test this hypothesis, we attempted to identify cell populations that possess osteoclast differentiation potential in the bone marrow, spleen, and blood by analyzing macrophage/monocyte-related cell surface markers such as CD11b, CD14, and colony-stimulating factor-1 receptor (c-Fms). In the bone marrow, the CD11b^? cell population, but not the CD11b⁺ cell population, differentiated into osteoclasts in the presence of receptor activator of nuclear factor-κB ligand and macrophage colony-stimulating factor. On the other hand, in the spleen and blood, CD11b⁺ cells differentiated into osteoclasts. Interestingly, lipopolysaccharide (LPS) administration to the mice dramatically increased the proportion of CD11b⁺ c-Fms⁺ CD14⁺ cells, which differentiated into osteoclasts, in the bone marrow and spleen. These results suggest that LPS administration increases the proportion of a distinct cell population expressing CD11b⁺, c-Fms⁺, and CD14⁺ in the bone marrow and spleen. Thus, these cell populations are considered to contribute to the increase in osteoclast number during inflammatory bone destruction such as periodontitis.     

Inhibitory effect of luteolin on osteoclast differentiation and function

Osteoclasts are multinucleated cells that play a crucial role in bone resorption, and are formed by the fusion of mononuclear osteoclasts derived from osteoclast precursors of the macrophage lineage. Compounds that specifically target functional osteoclasts would be ideal candidates for anti-resorptive agents for clinical applications. In the present study, we investigated the effects of luteolin, a flavonoid, on the regulation of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, functions and signaling pathway. Addition of luteolin to a coculture system of mouse bone marrow cells and ST2 cells in the presence of 10⁻⁸ M 1α,25(OH)₂D₃ caused significant inhibition of osteoclastogenesis. Luteolin had no effects on the 1α,25(OH)₂D₃-induced expressions of RANKL, osteoprotegerin and macrophage colony-stimulating factor mRNAs. Next, we examined the direct effects of luteolin on osteoclast precursors using bone marrow macrophages and RAW264.7 cells. Luteolin completely inhibited RANKL-induced osteoclast formation. Moreover, luteolin inhibited the bone resorption by mature osteoclasts accompanied by the disruption of their actin rings, and these effects were reversely induced by the disruption of the actin rings in mature osteoclasts. Finally, we found that luteolin inhibited RANKL-induced osteoclastogenesis through the suppression of ATF2, downstream of p38 MAPK and nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) expression, respectively. Taken together, the present results indicate that naturally occurring luteolin has inhibitory activities toward both osteoclast differentiation and functions through inhibition of RANKL-induced signaling pathway as well as actin ring disruption, respectively.     

Osteoclast precursors in bone marrow and peritoneal cavity

Osteoclasts differentiate from cells that share some phenotypes with mature macrophages and monocytes, but early precursors for osteoclasts still remain obscure. To characterize osteoclast precursors, using monoclonal anti-c-Fms and anti-c-Kit antibodies, bone marrow cells were separated and the frequency of clonogenic progenitors were measured. Osteoclast precursors in the bone marrow mainly expressed c-Kit and diminished in frequency when they expressed c-Fms. In contrast to bone marrow, the precursors in the peritoneal cavity were enriched with a population of c-Fms⁺. Injection of these antibodies into mice demonstrated that peritoneal osteoclast precursors were sensitive to anti-c-Fms but not to anti-c-Kit antibodies, whereas those in bone marrow only declined in the presence of both antibodies. Meanwhile, c-Fms as opposed to c-Kit played an essential role in the generation of osteoclasts in cultures. We also compared osteoclast precursors with colony forming cells (CFU-M) by a macrophage colony stimulating factor. CFU-M in bone marrow decreased when anti-c-Kit antibody was administered and no CFU-M was detected in peritoneum. In this study, we show differences between proliferative potential osteoclast precursors maintained in bone marrow and peritoneum and between CFU-M and osteoclast precursors. J. Cell. Physiol. 170:241–247, 1997. © 1997 Wiley-Liss, Inc.     

Distinct osteoclast precursors in the bone marrow and extramedullary organs characterized by responsiveness to Toll-like receptor ligands and TNF-alpha

Osteoclasts are derived from hemopoietic stem cells and play critical roles in bone resorption and remodeling. Multinucleated osteoclasts are attached tightly to bone matrix, whereas precursor cells with the potential to differentiate into osteoclasts in culture are widely distributed. In this study, we assessed the characteristics of osteoclast precursors in bone marrow (BM) and in extramedullary organs as indicated by their responsiveness to ligands for Toll-like receptors (TLRs) and to TNF-alpha. Development of osteoclasts from precursor cells in the BM was inhibited by CpG oligonucleotides, a ligand for TLR9, but not by LPS, a ligand for TLR4. BM osteoclasts were induced by TNF-alpha as well as receptor activator of NF-kappaB ligand in the presence of M-CSF. Splenic osteoclast precursors, even in osteoclast-deficient osteopetrotic mice, differentiated into mature osteoclasts following exposure to TNF-alpha or receptor activator of NF-kappaB ligand. However, splenic osteoclastogenesis was inhibited by both LPS and CpG. Osteoclastogenesis from peritoneal precursors was inhibited by not only these TLR ligands but also TNF-alpha. The effects of peptidoglycan, a ligand for TLR2, were similar to those of LPS. BM cells precultured with M-CSF were characterized with intermediate characteristics between those of splenic and peritoneal cavity precursors. Taken together, these findings demonstrate that osteoclast precursors are not identical in the tissues examined. To address the question of why mature osteoclasts occur only in association with bone, we may characterize not only the microenvironment for osteoclastogenesis, but also the osteoclast precursor itself in intramedullary and extramedullary tissues.     

Siglec-15, a member of the sialic acid-binding lectin, is a novel regulator for osteoclast differentiation

Osteoclasts are tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells derived from monocyte/macrophage-lineage precursors and are critically responsible for bone resorption. In giant cell tumor of bone (GCT), numerous TRAP-positive multinucleated giant cells emerge and severe osteolytic bone destruction occurs, implying that the emerged giant cells are biologically similar to osteoclasts. To identify novel genes involved in osteoclastogenesis, we searched genes whose expression pattern was significantly different in GCT from normal and other bone tumor tissues. By screening a human gene expression database, we identified sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) as one of the genes markedly overexpressed in GCT. The mRNA expression level of Siglec-15 increased in association with osteoclast differentiation in cultures of mouse primary unfractionated bone marrow cells (UBMC), RAW264.7 cells of the mouse macrophage cell line and human osteoclast precursors (OCP). Treatment with polyclonal antibody to mouse Siglec-15 markedly inhibited osteoclast differentiation in primary mouse bone marrow monocyte/macrophage (BMM) cells stimulated with receptor activator of nuclear factor κB ligand (RANKL) or tumor necrosis factor (TNF)-α. The antibody also inhibited osteoclast differentiation in cultures of mouse UBMC and RAW264.7 cells stimulated with active vitamin D₃ and RANKL, respectively. Finally, treatment with polyclonal antibody to human Siglec-15 inhibited RANKL-induced TRAP-positive multinuclear cell formation in a human OCP culture. These results suggest that Siglec-15 plays an important role in osteoclast differentiation.     

Connection between B lymphocyte and osteoclast differentiation pathways

Osteoclasts differentiate from the hemopoietic monocyte/macrophage cell lineage in bone marrow through cell-cell interactions between osteoclast progenitors and stromal/osteoblastic cells. Here we show another osteoclast differentiation pathway closely connected with B lymphocyte differentiation. Recently the TNF family molecule osteoclast differentiation factor/receptor activator of NF-kappaB ligand (ODF/RANKL) was identified as a key membrane-associated factor regulating osteoclast differentiation. We demonstrate that B-lymphoid lineage cells are a major source of endogenous ODF/RANKL in bone marrow and support osteoclast differentiation in vitro. In addition, B-lymphoid lineage cells in earlier developmental stages may hold a potential to differentiate into osteoclasts when stimulated with M-CSF and soluble ODF/RANKL in vitro. B-lymphoid lineage cells may participate in osteoclastogenesis in two ways: they 1) express ODF/RANKL to support osteoclast differentiation, and 2) serve themselves as osteoclast progenitors. Consistent with these observations in vitro, a decrease in osteoclasts is associated with a decrease in B-lymphoid cells in klotho mutant mice (KL(-/-)), a mouse model for human aging that exhibits reduced turnover during bone metabolism, rather than a decrease in the differentiation potential of osteoclast progenitors. Taken together, B-lymphoid lineage cells may affect the pathophysiology of bone disorders through regulating osteoclastogenesis.     

PPAR-gamma regulates osteoclastogenesis in mice

Osteoclasts are bone-resorbing cells derived from hematopoietic precursors of the monocyte-macrophage lineage. Regulation of osteoclast function is central to the understanding of bone diseases such as osteoporosis, rheumatoid arthritis and osteopetrosis. Although peroxisome proliferator-activated receptor-gamma (PPAR-gamma) has been shown to inhibit osteoblast differentiation, its role, if any, in osteoclasts is unknown. This is a clinically crucial question because PPAR-gamma agonists, "such as thiazolidinediones-" a class of insulin-sensitizing drugs, have been reported to cause a higher rate of fractures in human patients. Here we have uncovered a pro-osteoclastogenic effect of PPAR-gamma by using a Tie2Cre/flox mouse model in which PPAR-gamma is deleted in osteoclasts but not in osteoblasts. These mice develop osteopetrosis characterized by increased bone mass, reduced medullary cavity space and extramedullary hematopoiesis in the spleen. These defects are the result of impaired osteoclast differentiation and compromised receptor activator of nuclear factor-kappaB ligand signaling and can be rescued by bone marrow transplantation. Moreover, ligand activation of PPAR-gamma by rosiglitazone exacerbates osteoclast differentiation in a receptor-dependent manner. Our examination of the underlying mechanisms suggested that PPAR-gamma functions as a direct regulator of c-fos expression, an essential mediator of osteoclastogenesis. Therefore, PPAR-gamma and its ligands have a previously unrecognized role in promoting osteoclast differentiation and bone resorption.     

Temporal and spatial localization of osteoclasts in colonies from embryonic stem cells

Osteoclasts are hematopoietic cells essential for bone resorption. To understand the process of osteoclastogenesis, we have developed a culture system that employs a stromal cell line, in which differentiation of osteoclasts from single embryonic stem (ES) cells occurs. This culture, which did not require any cell passaging or other manipulations, enabled us to investigate the temporal and spatial localization of the osteoclast lineage in the colonies formed from ES cells. Cells expressing tartrate-resistant acid phosphatase, a specific marker of the osteoclast lineage, were first detected on day 8, and subsequently became localized at the periphery of colonies and matured into multinucleated cells to resorb bone. Addition of macrophage colony-stimulating factor and osteoprotegerin-ligand, which are produced by stromal cells, promoted osteoclastogenesis in whole colonies, indicating that the location and maintenance of mature osteoclasts as well as the growth and differentiation of osteoclast precursors are regulated by these two factors.     

A novel osteoclast precursor cell line, 4B12, recapitulates the features of primary osteoclast differentiation and function: Enhanced transfection efficiency before and after differentiation

Osteoclasts are bone‐resorbing multinucleated cells differentiated from monocyte/macrophage lineage precursors. A novel osteoclast precursor cell line, 4B12 was established from Mac‐1⁺c‐Fms⁺RANK⁺ cells from calvaria of 14‐day‐old mouse embryos using immunofluorescence and cell‐sorting methods. Like M‐CSF‐dependent bone marrow macrophages (M‐BMMs), M‐CSF is required for 4B12 cells to differentiate into TRAP‐positive multinucleated cells [TRAP(+) MNCs] in the presence of RANKL. Bone‐resorbing osteoclasts differentiated from 4B12 cells on dentine slices possess both a clear zone and ruffled borders and express osteoclast‐specific genes. Bone‐resorbing activity, but not TRAP, was enhanced in the presence of IL‐1α. The number of TRAP(+) MNCs and the number of pits formed from 4B12 cells on dentine slices was fourfold higher than that from M‐BMMs. 4B12 cells were identified as macrophages with Mac‐1 and F4/80, yet lost these markers upon differentiation into osteoclasts as determined by confocal laser scanning microscopy. The 4B12 cells do not have the potential to differentiate into dendritic cells indicating commitment to the osteoclast lineage. 4B12 cells are readily transfectable with siRNA transfection before and after differentiation. These data show that 4B12 cells faithfully replicate the properties of primary cells and are a useful and powerful model for analyzing the molecular and cellular regulatory mechanisms of osteoclastogenesis and osteoclast function. J. Cell. Physiol. 221: 40–53, 2009. © 2009 Wiley‐Liss, Inc     

Increasing membrane-bound MCSF does not enhance OPGL-driven osteoclastogenesis from marrow cells

Macrophage colony-stimulating factor (MCSF) and osteoprotegerin ligand (OPGL), both produced by osteoblasts/stromal cells, are essential factors for osteoclastogenesis. Whether local MCSF levels regulate the amount of osteoclast formation is unclear. Two culture systems, ST-2 and Chinese hamster ovary-membrane-bound MCSF (CHO-mMCSF)-Tet-OFF cells, were used to study the role of mMCSF in osteoclast formation. Cells from bone marrow (BMM) or spleen were cultured with soluble OPGL on glutaraldehyde-fixed cell layers; osteoclasts formed after 7 days. Osteoclast number was proportional to the amount of soluble OPGL added. In contrast, varying mMCSF levels in the ST-2 or CHO-mMCSF-Tet-OFF cell layers, respectively by variable plating or by addition of doxycycline, did not affect BMM osteoclastogenesis: 20-450 U of mMCSF per well generated similar osteoclast numbers. In contrast, spleen cells were resistant to mMCSF: osteoclastogenesis required > or = 250 U per well and further increased as mMCSF rose higher. Our results demonstrate that osteoclast formation in the local bone environment is dominated by OPGL. Increasing mMCSF above basal levels does not further enhance osteoclast formation from BMMs, indicating that mMCSF does not play a dominant regulatory role in the bone marrow.     

Cadherin-6 Mediates the Heterotypic Interactions between the Hemopoietic Osteoclast Cell Lineage and Stromal Cells in a Murine Model of Osteoclast Differentiation

Osteoclasts are multinucleated cells of hemopoietic origin that are responsible for bone resorption during physiological bone remodeling and in a variety of bone diseases. Osteoclast development requires direct heterotypic cell–cell interactions of the hemopoietic osteoclast precursors with the neighboring osteoblast/stromal cells. However, the molecular mechanisms underlying these heterotypic interactions are poorly understood. We isolated cadherin-6 isoform, denoted cadherin-6/2 from a cDNA library of human osteoclast-like cells. The isolated cadherin-6/2 is 3,423 bp in size consisting of an open reading frame of 2,115 bp, which encodes 705 amino acids. This isoform lacks 85 amino acids between positions 333 and 418 and contains 9 different amino acids in the extracellular domain compared with the previously described cadherin-6. The human osteoclast-like cells also expressed another isoform denoted cadherin-6/1 together with the cadherin-6. Introduction of cadherin-6/2 into L-cells that showed no cell–cell contact caused evident morphological changes accompanied with tight cell–cell association, indicating the cadherin-6/2 we isolated here is functional. Moreover, expression of dominant-negative or antisense cadherin-6/2 construct in bone marrow–derived mouse stromal ST2 cells, which express only cadherin-6/2, markedly impaired their ability to support osteoclast formation in a mouse coculture model of osteoclastogenesis. Our results suggest that cadherin-6 may be a contributory molecule to the heterotypic interactions between the hemopoietic osteoclast cell lineage and osteoblast/bone marrow stromal cells required for the osteoclast differentiation. Since both osteoclasts and osteoblasts/bone marrow stromal cells are the primary cells controlling physiological bone remodeling, expression of cadherin-6 isoforms in these two cell types of different origin suggests a critical role of these molecules in the relationship of osteoclast precursors and cells of osteoblastic lineage within the bone microenvironment.     

The phosphatidylinositol 3-kinase-mediated production of interferon-beta is critical for the lipopolysaccharide inhibition of osteoclastogenesis

AIMS: This study was performed to define molecular mechanisms underlying lipopolysaccharide (LPS)-induced inhibition of osteoclastogenesis. MAIN METHODS: The LPS-dependent signaling pathways leading to the inhibition of osteoclastogenesis were examined using chemical inhibitors and neutralizing antibodies. Key findings: Lipopolysaccharide (LPS) has been shown to induce massive bone loss in vivo by stimulating osteoclast differentiation and activity. However, the direct effect of LPS on osteoclastogenesis in vitro is complex. Based on the context of the differentiation state of precursors, LPS has been shown to either augment or inhibit osteoclast differentiation. When added to receptor activator of NF-kappaB ligand (RANKL)-primed precursors, LPS enhances osteoclast differentiation via the production of TNF-alpha. On the other hand, LPS inhibits osteoclastogenesis from early precursors like bone marrow macrophages by hitherto unknown mechanism. In the present study, we investigated the mechanism by which LPS inhibits osteoclastogenesis. We have identified that the phosphatidylinositol 3-kinase (PI 3-kinase) dependent production of IFN-beta and resultant inhibition of c-Fos expression upon LPS stimulation of bone marrow macrophages are responsible for the LPS-induced inhibition of osteoclastogenesis. Inhibition of PI 3-kinase, neutralization of IFN-beta, and overexpression of c-Fos respectively prevented the LPS-induced inhibition of osteoclast differentiation. SIGNIFICANCE: Our results provide a molecular understanding of the differentiation stage-specific dual effect of LPS on osteoclastogenesis.     

High mobility group box 1 protein regulates osteoclastogenesis through direct actions on osteocytes and osteoclasts in vitro

Old age and Cx43 deletion in osteocytes are associated with increased osteocyte apoptosis and osteoclastogenesis. We previously demonstrated that apoptotic osteocytes release elevated concentrations of the proinflammatory cytokine, high mobility group box 1 protein (HMGB1) and apoptotic osteocyte conditioned media (CM) promotes osteoclast differentiation. Further, prevention of osteocyte apoptosis blocks osteoclast differentiation and attenuates the extracellular release of HMGB1 and RANKL. Moreover, sequestration of HMGB1, in turn, reduces RANKL production/release by MLO-Y4 osteocytic cells silenced for Cx43 (Cx43^def), highlighting the possibility that HMGB1 promotes apoptotic osteocyte-induced osteoclastogenesis. However, the role of HMGB1 signaling in osteocytes has not been well studied. Further, the mechanisms underlying its release and the receptor(s) responsible for its actions is not clear. We now report that a neutralizing HMGB1 antibody reduces osteoclast formation in RANKL/M-CSF treated bone marrow cells. In bone marrow macrophages (BMMs), toll-like receptor 4 (TLR4) inhibition with LPS-RS, but not receptor for advanced glycation end products (RAGE) inhibition with Azeliragon attenuated osteoclast differentiation. Further, inhibition of RAGE but not of TLR4 in osteoclast precursors reduced osteoclast number, suggesting that HGMB1 produced by osteoclasts directly affects differentiation by activating TLR4 in BMMs and RAGE in preosteoclasts. Our findings also suggest that increased osteoclastogenesis induced by apoptotic osteocytes CM is not mediated through HMGB1/RAGE activation and that direct HMGB1 actions in osteocytes stimulate pro-osteoclastogenic signal release from Cx43^def osteocytes. Based on these findings, we propose that HMGB1 exerts dual effects on osteoclasts, directly by inducing differentiation through TLR4 and RAGE activation and indirectly by increasing pro-osteoclastogenic cytokine secretion from osteocytes.