Osteoclast Formation In Cocultures Research Articles

Magnolol Inhibits Osteoclast Differentiation via Suppression of RANKL Expression

Magnolol, a compound from the traditional Korean herb Magnolia sp., has been exhaustively investigated as a therapeutic agent against several diseases including systemic and local inflammation. We examined the effects of magnolol on osteoclastic differentiation associated with inflammation. Magnolol markedly reduced interleukin (IL)-1-induced osteoclast formation in co-cultures of murine osteoblasts and bone marrow cells, whereas it had no effect on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation in bone marrow macrophage cultures. In osteoblasts, magnolol markedly inhibited both the up-regulation of RANKL expression and the production of prostaglandin E2 (PGE2) in response to IL-1 treatment. Addition of exogenous PGE2 reversed the inhibitory effects of magnolol on IL-1-induced RANKL expression in osteoblasts and osteoclast formation in co-cultures. Magnolol inhibited IL-1-induced PGE2 production, at least in part by suppressing cyclooxygenase-2 (COX-2) expression. Taken together, these results demonstrate that magnolol inhibits IL-1-induced RANKL expression in osteoblasts through suppression of COX-2 expression and PGE2 production, resulting in inhibition of osteoclast differentiation in co-cultures.

Indoxyl sulfate, a uremic toxin in chronic kidney disease, suppresses both bone formation and bone resorption.

Abnormalities of bone turnover are commonly observed in patients with chronic kidney disease (CKD), and the low‐turnover bone disease is considered to be associated with low serum parathyroid hormone (PTH) levels and skeletal resistance to PTH. Indoxyl sulfate (IS) is a representative uremic toxin that accumulates in the blood of patients with CKD. Recently, we have reported that IS exacerbates low bone turnover induced by parathyroidectomy (PTX) in adult rats, and suggested that IS directly induces low bone turnover through the inhibition of bone formation by mechanisms unrelated to skeletal resistance to PTH. To define the direct action of IS in bone turnover, we examined the effects of IS on bone formation and bone resorption in vitro. In cultures of mouse primary osteoblasts, IS suppressed the expression of osterix, osteocalcin, and bone morphogenetic protein 2 (BMP2) mRNA and clearly inhibited the formation of mineralized bone nodules. Therefore, IS directly acts on osteoblastic cells to suppress bone formation. On the other hand, IS suppressed interleukin (IL)‐1‐induced osteoclast formation in cocultures of bone marrow cells and osteoblasts, and IL‐1‐induced bone resorption in calvarial organ cultures. In cultures of osteoblasts, IS suppressed the mRNA expression of RANKL, the receptor activator of NF‐κB ligand, which is a pivotal factor for osteoclast differentiation. Moreover, IS acted on osteoclast precursor, bone marrow‐derived macrophages and RAW264.7 cells, and suppressed RANKL‐dependent differentiation into mature osteoclasts. IS may induce low‐turnover bone disease in patients with CKD by its direct action on both osteoblasts and osteoclast precursors to suppress bone formation and bone resorption.

Rosmarinic acid exerts an antiosteoporotic effect in the RANKL-induced mouse model of bone loss by promotion of osteoblastic differentiation and inhibition of osteoclastic differentiation.

Bone homeostasis is ensured by the balance between bone formation and resorption. Thus, control of the recruitment, proliferation, and differentiation of bone cells is essential to maintain bone mass. The aim of this study was to elucidate the effects of rosmarinic acid as a potential therapeutic agent on bone metabolism using bone cells and a mouse model. Rosmarinic acid increased alkaline phosphatase activity and induced mineralization in osteoblasts. Addition of rosmarinic acid to cultures of calvarial osteoblastic cells prepared from T-cell factor/β-catenin TOP-GAL mutant mice strongly induced the expression of LacZ and promoted stabilization of β-catenin in the cytoplasm of ST2 cells, suggesting that rosmarinic acid affects the canonical Wnt signaling pathway. Moreover, rosmarinic acid inhibited not only osteoclast formation in cocultures of mouse bone marrow cells and osteoblasts, but also receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastic differentiation in bone marrow-derived macrophages. RANKL-induced p38 mitogen-activated protein kinase and the expression of nuclear factor of activated T cell, c-Jun, and c-Fos were inhibited by rosmarinic acid in bone marrow macrophages. Finally, we confirmed that rosmarinic acid improved bone mass in a soluble RANKL-induced bone loss mouse model. Rosmarinic acid has dual regulatory effects on bone metabolism and may control the bone functions by controlling osteoblastic and osteoclastic differentiation.

Trapidil, a platelet-derived growth factor antagonist, inhibits osteoclastogenesis by down-regulating NFATc1 and suppresses bone loss in mice

Trapidil, a platelet-derived growth factor antagonist, was originally developed as a vasodilator and anti-platelet agent and has been used to treat patients with ischemic coronary heart, liver, and kidney disease. In this study, we investigated the effects of trapidil on osteoclastogenesis and elucidated the possible mechanism of action of trapidil. Trapidil strongly inhibited osteoclast formation in co-cultures of bone marrow cells and osteoblasts without affecting receptor activator of NF-κB ligand (RANKL) or osteoprotegerin expression in osteoblasts. In addition, trapidil suppressed RANKL-induced osteoclast formation from osteoclast precursors. Trapidil reduced RANKL-induced expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), a master transcription factor for osteoclastogenesis, without affecting the expression of c-Fos that functions as a key upstream activator of NFATc1 during osteoclastogenesis. Ectopic expression of a constitutively active form of NFATc1 reversed the anti-osteoclastogenic effect of trapidil, indicating that NFATc1 is a critical target of the anti-osteoclastogenic action of trapidil. RANKL-induced calcium oscillation and Pim-1 expression, which are required for NFATc1 induction and osteoclastogenesis, were abrogated by trapidil. Consistent with the in vitro results, trapidil had a potent inhibitory effect on osteoclast formation and bone resorption induced by interleukin-1 in an animal model. Taken together, our data demonstrate that trapidil abrogates RANKL-induced calcium oscillation and Pim-1 expression required for NFATc1 induction, thereby inhibiting osteoclastogenesis.

破骨細胞分化に対するマクロファージ遊走阻止因子(MIF)の影響について

破骨細胞分化に対するマクロファージ遊走阻止因子(MIF)の影響について

The Protective Effects of β-Cryptoxanthin on Inflammatory Bone Resorption in a Mouse Experimental Model of Periodontitis

We examined the effects of β-cryptoxanthin, a typical carotenoid, on inflammatory periodontitis. β-Cryptoxanthin suppressed lipopolysaccharide (LPS)-induced osteoclast formation in co-cultures of bone marrow cells and osteoblasts. In a mouse model of periodontitis, it suppressed bone resorption in the mandibular alveolar bone in vitro and restored alveolar bone loss induced by LPS in vivo. β-Cryptoxanthin might protect against periodontal disease.

Effects of veraguensin and galgravin on osteoclast differentiation and function

The dried flower buds of Magnolia sp. are widely used as herbal medicines because of their anti-inflammatory, anti-malarial and anti-platelet activities. Here, we found that veraguensin and galgravin, lignan compounds derived from Magnolia sp., dose-dependently inhibited osteoclast formation in co-cultures of bone marrow cells and osteoblastic cells. These compounds also inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation in RAW264.7 cells and bone marrow macrophages. In the RANKL-induced signaling pathway, veraguensin and galgravin reduced p38 phosphorylation and suppressed the expression of c-Fos, a key transcription factor for osteoclastogenesis. Veraguensin and galgravin also inhibited osteoclastic pit formation, which was accompanied by decreased mature osteoclast viability. In conclusion, these results indicate that veraguensin and galgravin can inhibit bone resorption and may offer novel compounds for the development of drugs to treat bone-destructive diseases such as osteoporosis.

α-Tocopheryl Succinate Inhibits Osteoclast Formation by Suppressing Receptor Activator of Nuclear Factor-kappaB Ligand (RANKL) Expression and Bone Resorption

ObjectiveOsteoclasts are bone-resorbing multinucleated cells derived from the monocyte/macrophage lineage during normal and pathological bone turnover. Recently, several studies revealed that alpha-tocopheryl succinate (αTP-suc) have demonstrated potent anti-cancer activities in vitro and in vivo. However, the effects of αTP-suc on osteoclast formation and bone resorption remain unknown. Thus, in this study, we examined the effects of αTP-suc on osteoclast differentiation and bone resorbing activity in inflammatory bone loss model.MethodsOsteoclast differentiation assay was performed by cocultures of mouse bone marrow cells and calvarial osteoblasts in culture media including interleukin-1 (IL-1). Osteoclasts were stained for tartrate-resistant acid phosphatase (TRAP). The level of receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). ICR mice were administered an intraperitoneal injections of αTP-suc or dimethyl sulfoxide (DMSO) 1 day before the implantation of a freeze-dried collagen sponge loaded with phosphate-buffered saline (PBS) or IL-1 over the calvariae and every other day for 7 days. The whole calvariae were obtained and analyzed by micro-computed tomography (CT) scanning, and stained for TRAP.ResultsαTP-suc inhibits osteoclast formation in cocultures stimulated by IL-1 and decreased the level of expression of RANKL mRNA in osteoblasts. In addition, administered intraperitoneal injections of αTP-suc prevented IL-1-mediated osteoclast formation and bone loss in vivo.ConclusionOur findings suggest that αTP-suc may have therapeutic value for treating and preventing bone-resorptive diseases, such as osteoporosis.

The ZZ Domain of Sequestosome-1/p62 Plays An Important Role In Stromal Cell Support of Myeloma Cell Growth and Osteoclast Formation

AbstractAbstract 128The marrow microenvironment provides a critical supportive role in myeloma (MM) and enhances both tumor growth and bone destruction through activation of multiple signaling pathways in stromal cells. We reported that sequestosome 1 (p62) plays a key role in the formation of signaling complexes that result in NF-kB, p38 MAPK, and PI3K activation in the marrow microenvironment of patients with MM. These results suggest that p62 is a potential therapeutic target for blocking the supportive effects of the marrow microenvironment in MM. The goal of this study was to identify the domains of p62 responsible for increased MM cell growth and osteoclast (OCL) formation mediated by NF-kB and p38 MAPK signaling, as a means to develop inhibitory peptides/molecules as potential therapeutic agents for MM. To pursue this objective, we generated deletion constructs of p62 that lacked specific p62 domains: ΔSH2, ΔPB1, ΔZZ, Δp38, ΔTBS and ΔUBA domains. We then transfected these constructs into a p62-knockout (KO) stromal cell-line we established from p62-KO mice and examined their RANKL, IL-6, and VCAM-1 expression induced by TNF-a. GFP-labeled MM1.S myeloma cells or normal CFU-GM, a source of OCL precursors, were then co-cultured with the p62-KO cells transduced with the different p62 deletion constructs and compared to wild type (WT) stromal cells. IL-6 production and VCAM-1 expression induced by TNF-a was 50% lower in non-transduced p62-KO stromal cells compared to WT stromal cells. Further, in contrast to WT cells, RANKL was not induced by TNF-a in the p62-KO stromal cell-line, and OCL formation in co-cultures of p62-KO stromal cells with CFU-GM was very low. Transduction of p62-KO stromal cells with the ΔSH2, ΔPB1, Δp38 and ΔUBA constructs restored stromal cell support of MM growth, VCAM-1 and IL-6 production. However, p62-KO stromal cells transduced with the ΔZZ construct did not increase MM cell growth, or increase IL-6 and VCAM-1 expression, or fully restore the capacity of the p62-KO stromal cells to support OCL formation. These results demonstrate that the ZZ domain of p62 is required for stromal cell support of MM cell growth, increased IL-6 and VCAM-1 expression, and OCL formation. These results suggest that dominant negative constructs or small molecules that target the ZZ domain of p62 should block p62 function and inhibit support of MM cells and OCL formation by the marrow microenvironment. Disclosures:Roodman:Amgen, Celgene, Acceleron & Millennium: Consultancy.

Comparison between MLO-Y4 osteocyte and osteoblast to support osteoclast formation in vitro

To compare between MLO-Y4 osteocyte and osteoblast to support osteoclast formation in co-culture system. MLO-Y4 cells and murine osteoblast cells were co-cultured with bone marrow cells with or without vitamin D₃ presence.Bone marrow cells were as control group. Tartrat resistant acid phosphatase (TRAP)+ giant cells with three or more nuclei were counted and compared under a microscope at day 9. In the absence of vitamin D₃, (1963.3 ± 93.1)/plate osteoclasts were observed when MLO-Y4 cells co-cultured with bone marrow cells in 24-well plate.While only (12.7 ± 5.5)/plate osteoclasts were found in the osteoblast group, and (6.0 ± 1.0)/plate in control group. The statistical difference occurs for any two groups (P < 0.05). Vitamin D₃ could significantly increase osteoclast formation in the three groups. Osteocytes could induce osteoclastogenesis without the presence of vitamin D₃ and vitamin D₃ could enhance the induction effects of MLO-Y4 and osteoblast cells.

Green tea polyphenol (−)‐epigallocatechin gallate suppressed the differentiation of murine osteoblastic MC3T3‐E1 cells

Recently, various physiological effects of the tea polyphenol catechin for alleviating diseases such as cancer, arteriosclerosis, hyperlipidaemia and osteoporosis have been reported. However, the physiological effect of catechin on bone metabolism remains unclear. We examined the physiological effect of EGCG [(-)-epigallocatechin-3-gallate], which is the main component of green tea catechin, on osteoblast development using the precursor cell line of osteoblasts, MC3T3-E1, and co-culture of the osteoblasts from mouse newborn calvaria and mouse bone marrow cells. Although EGCG did not affect the viability and proliferation of MC3T3-E1 cells, EGCG inhibited the osteoblast differentiation. Furthermore, EGCG did not affect the mineralization of differentiated MC3T3-E1 cells, and reduced osteoclast formation in co-culture. These results suggest that EGCG can effectively suppress bone resorption, and can be used as an effective medicine in the treatment of the symptoms of osteoporosis.

Trichostatin A inhibits osteoclastogenesis and bone resorption by suppressing the induction of c-Fos by RANKL

Histone deacetylases are enzymes involved in the remodeling of chromatin structure, in the regulation of transcriptional activity, and in epigenetic integrity. Histone deacetylase inhibitors such as trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) have emerged as potent anticancer drugs that have proved useful in preclinical and early clinical trials. The role of histone deacetylase inhibitors in regulating osteoclast differentiation, however, is not well established. In this study, we analyzed the effects of TSA on osteoclast differentiation induced by the differentiation factor RANKL (receptor activator of NF-κB ligand). TSA strongly inhibited osteoclast formation in coculture of bone marrow cells and osteoblasts without reducing RANKL expression in osteoblasts. Furthermore, TSA suppressed RANKL-induced osteoclast formation from primary bone marrow-derived macrophages. TSA was only effective when present during the early stage of osteoclast differentiation. This effect was accompanied by a significant decrease in the RANKL-stimulated induction of c-Fos and NFATc1, which are key transcription factors during early osteoclastogenesis. The ectopic introduction of c-Fos and a constitutively active form of NFATc1 reversed the TSA-induced antiosteoclastogenic effect. Consistent with the in vitro results, TSA inhibited lipopolysaccharide- and interleukin-1-induced bone resorption and osteoclast formation in an in vivo model. Taken together, our findings suggest a novel action of TSA: inhibiting RANKL-induced osteoclast formation by suppressing the induction of the osteoclastogenic transcription factor c-Fos. Also, the inhibitory effect of TSA on bone destruction in vivo suggests that histone deacetylase inhibitors may be novel therapeutics for treating typical bone diseases.

Phosphodiesterase 3 and 4 Negatively Regulate Receptor Activator of Nuclear Factor-.KAPPA.B Ligand-Mediated Osteoclast Formation by Prostaglandin E2

Prostaglandin E2 (PGE2) stimulates osteoclast formation by increasing receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) mRNA expression via cAMP-protein kinase A (PKA) pathways in osteoblasts. Since phosphodiesterases (PDEs) balance the concentration of intracellular cAMP stimulated by PGE2, we investigated the role of PDEs in PGE2-mediated osteoclast formation using various cAMP-specific PDEs inhibitors. In the presence of PGE(2), PDE3 and 4 inhibitors were shown to dose-dependently increase the osteoclast formation in cocultures of mouse bone marrow cells and calvarial osteoblasts. In agreement with this finding, they stimulated PGE2-induced cAMP production followed by increased RANKL mRNA expression in osteoblasts, suggesting that PDE3 and 4 negatively regulate PGE2-mediated RANKL expression in osteoblasts. RT-PCR analysis revealed that PDE3A, 3B, 4A, 4B and 4D are expressed in osteoblasts. The PDE8 inhibitor did not increase osteoclast formation, although it stimulated PGE2-induced RANKL mRNA expression in osteoblasts. The four subtypes of PGE receptors are designated EP1, EP2, EP3, and EP4. PDE3 and 4 inhibitors were found to increase EP1/3, EP4 and/or EP2 agonist-stimulated RANKL expression, indicating that PDE3 and PDE4 negatively regulate PGE2-induced RANKL mRNA expression through four EPs. Taken together, these data suggest that PDE3 and PDE4 could have important pharmacological and clinical implications in bone-related diseases.

Adrenomedullin inhibits MAPK pathway-dependent rheumatoid synovial fibroblast-mediated osteoclastogenesis by IL-1 and TNF-α

The objective of this study is to determine the effects of adrenomedullin (AM) on IL-1- and TNF-alpha-induced rheumatoid synovial fibroblasts (RASFs)-mediated osteoclastogenesis. The formation of osteoclasts in co-cultures of RASFs and peripheral blood mononuclear cells was evaluated by tartrate-resistant acid phosphatase and resorption pit formation assay. The expression of RANKL, OPG, p-ERK, p-p38, and p-JNK was examined by immunoblotting and quantitative reverse transcription-polymerase chain reaction. AM (1-52) inhibits IL-1- and TNF-alpha-induced RASFs-mediated osteoclastogenesis. AM affected IL-1-, TNF-alpha-induced RANKL and OPG expression in RASFs. AM also inhibits IL-1 and TNF-alpha-induced phosphorylation of ERK-1/2, p38 MAPK, and JNK. Inhibitor of AM (AM 22-52) inhibits the effects of AM on the osteoclastogenesis. These results suggest that AM might be involved in the inflammatory cytokines-mediated osteoclastogenesis and thus bone damage, and indicate that it can be a new therapeutic strategy against joint destruction in RA.

Suppressive effects of Anoectochilus formosanus extract on osteoclast formation in vitro and bone resorption in vivo

Anoectochilus formosanus, a plant native to Taiwan, is used as a folk medicine. It was found that oral administration of A. formosanus extract (AFE) (500 mg/kg) for 4 weeks suppressed bone weight loss and trabecular bone loss in ovariectomized mice, an experimental model of osteoporosis. Although AFE at 12.5 and 25 mug/ml inhibited osteoclast formation in co-culture of osteoblasts and bone marrow cells, AFE did not inhibit the formation of osteoclast progenitor cells and preosteoclast cells in bone marrow cells and RAW264 cells. However, AFE (at 12.5 and 25 microg/ml) decreased RANKL expression. These results suggested that AFE might suppress the bone loss caused by estrogen deficiency through suppression of RANKL expression required for osteoclast formation.

Rolipram, a phosphodiesterase 4 inhibitor, suppresses PGE2-induced osteoclast formation by lowering osteoclast progenitor cell viability

We have previously shown that phosphodiesterase (PDE) inhibitors induce osteoclast formation by suppressing the degradation of intracellular cAMP. To determine the regulatory roles of PDE inhibitors on PGE2-induced osteoclastogenesis, we investigated the effect of PDE inhibitors on osteoclast formation in the presence of PGE2. We found that IBMX, a nonselective PDE inhibitor, and rolipram, a specific PDE4 inhibitor, decreased PGE2-induced osteoclast formation in cocultures of mouse bone marrow cells and osteoblastic cells. These suppressive effects were observed only when cocultures were treated with PDE inhibitors in the presence of PGE2 at an early stage of differentiation. Northern blot analysis revealed that the PDE4 inhibitor works synergistically with PGE2 to increase the ratio of TRANCE/OPG mRNA in osteoblasts, suggesting that suppression of osteoclast formation by PGE2 and the PDE4 inhibitor is not attributable to their indirect effect on calvarial osteoblasts. We further demonstrated that the PDE4 inhibitor augments the inhibitory effect of PGE2 on osteoclast progenitor cell viability, showing that combined treatment with PGE2 and rolipram suppresses osteoclast formation by directly reducing osteoclast progenitor cell viability.

Rheumatoid synovial endothelial cells produce macrophage colony-stimulating factor leading to osteoclastogenesis in rheumatoid arthritis

Periarticular osteoporosis and joint destruction are major complications in rheumatoid arthritis (RA), caused by osteoclast-mediated bone resorption. However, the mechanisms of monocyte/osteoclast maturation and role of RA endothelial cells (RAECs) in the control of osteoclastogenesis remain unclear. The present study was designed to determine the most important factors that influence monocyte accumulation and osteoclast formation among the many factors produced by RAEC. We analysed the expression profiles of various genes in human endothelial cells from various organs (RA synovium, umbilical vein, skin, liver sinusoid, renal glomerulus and brain) using oligonucleotide microarrays. Specifically, up-regulated gene in RAECs was assessed by real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay and immunostaining of RA synovia. Migration of monocytes was assessed by the chemotactic chamber EZ-TAXIScan. Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cell (MNC) formation was observed by microscopy. Among many epithelial-expressed factors, macrophage colony-stimulating factor (M-CSF) gene was abundantly expressed specifically in RAECs. Genes of fibroblast growth factor-2, interleukin-6 and osteoprotegerin were also overexpressed in RAECs. Migration of monocytes and osteoclast formation in co-cultures promoted by culture supernatants of RAECs were inhibited by M-CSF neutralizing antibody. M-CSF produced by RAECs is involved in osteoclastogenesis from monocytes, migration and TRAP-positive MNC formation.

CD40 ligation of rheumatoid synovial fibroblasts regulates RANKL‐medicated osteoclastogenesis: Evidence of NF‐κB–dependent, CD40‐mediated bone destruction in rheumatoid arthritis

To determine whether CD40 ligation of rheumatoid arthritis synovial fibroblasts (RASFs) is able to induce RANKL expression and osteoclastogenesis in RASFs, and to identify its mechanism of action in patients with RA. CD40 of RASFs was ligated with CD40 ligand (CD40L)-transfected L cells or activated T cells. The formation of osteoclasts in cocultures of CD40-ligated RASFs and T lymphocyte-depleted peripheral blood mononuclear cells was evaluated by tartrate-resistant acid phosphatase staining, detection of calcitonin receptor, and resorption pit formation assay. The expression of NF-kappaB, IkappaB alpha, ERK-1/2, phospho-ERK-1/2, p38, phospho-p38, and RANKL was examined by immunoblotting and/or semiquantitative reverse transcription-polymerase chain reaction. CD40 ligation of RASFs by CD40L-transfected L cells or activated T cells induced RANKL expression and enhanced osteoclastogenesis. CD40 ligation of RASFs also induced activation of ERK-1/2, p38 MAPK, and NF-kappaB and up-regulation of CD40 ligation-induced RANKL expression, whereas osteoclastogenesis was reduced in RASFs transfected with a dominant-negative mutant of IkappaB alpha or by an NF-kappaB inhibitor. However, specific inhibitors of MAPK/ERK-1/2 and p38 MAPK partially blocked the induction of RANKL expression and osteoclastogenesis. Monoclonal antibodies against interleukin-1 and tumor necrosis factor alpha partially inhibited CD40 ligation-mediated osteoclastogenesis. These results indicate that CD40 ligation of RASFs induces RANKL expression mainly via NF-kappaB activation and also results in enhanced osteoclast formation, both of which might play important roles in bone and cartilage destruction in RA. Inhibition of the CD40-CD40L interaction is a potential strategy for the prevention of bone damage in RA.

Bone stromal cells in pagetic bone and Paget's sarcoma express RANKL and support human osteoclast formation

Paget's disease is a focal disorder of bone remodelling, in which there is an increase in osteoclast formation. A rare complication of Paget's disease is the development of a sarcoma, most commonly an osteosarcoma. Osteoclast formation occurs in the presence of macrophage-colony stimulating factor and receptor activator for nuclear factor-kappaB ligand (RANKL), and it has been shown that bone stromal cells in Paget's disease can influence osteoclast formation by modulating the expression of RANKL and its decoy receptor, osteoprotegerin (OPG). In this study we show that pagetic bone stromal cells express RANKL and that these cells promote osteoclast formation by a RANKL-dependent mechanism. Osteoclast formation in co-cultures of monocytes and either pagetic bone stromal cells or Paget's sarcoma stromal cells was not only induced by a contact-dependent mechanism but also occurred via the release of a soluble factor. In contrast to bone stromal cells isolated from normal controls, stromal cells isolated from morphologically normal bone in one patient with Paget's disease also stimulated osteoclast formation in this way; this osteoclastogenesis was inhibited by OPG. Our results indicate that Paget's bone stromal cells support osteoclast formation by a RANKL-dependent process which involves not only cell-cell contact but also secretion of soluble RANKL.

RANK ligand expression in heat shock factor‐2 deficient mouse bone marrow stromal/preosteoblast cells

Heat Shock Proteins (HSP) are molecular chaperones activated upon cellular stress/stimuli. HSP gene expression is regulated by Heat Shock Factors (HSF). We have recently demonstrated a functional role for heat shock factor-2 (HSF-2) in fibroblast growth factor-2 (FGF-2)-induced RANK ligand (RANKL), a critical osteoclastogenic factor expression on stromal/preosteoblast cells. In the present study, we show that FGF-2 treatment did not induce RANKL expression in HSF-2-/-stromal/preosteoblast cells. Interestingly, HSF-2 deficiency resulted in rapid induction of alkaline phosphatase (ALP) activity and osteocalcin mRNA expression in these cells. Furthermore, FGF-2 did not induce osteoclast formation in co-culture of normal mouse spleen cells and HSF-2-/-stromal/preosteoblast cells. Electron microscopy analysis demonstrated that osteoclasts from HSF-2-/-mice have poorly developed ruffled borders. These data further confirm that HSF-2 plays an important role in FGF-2-induced RANKL expression in stromal/preosteoblast cells. HSF-2 deficiency has pleotropic effects on gene expression during osteoblast differentiation and osteoclastogenesis in the bone microenvironment. Novel therapeutic agents that modulate HSF-2 activation may have therapeutic utility against increased levels of FGF-2 and bone destruction associated with pathologic conditions.