Osteoclast Formation In Cocultures Research Articles

α-Lipoic Acid Inhibits Inflammatory Bone Resorption by Suppressing Prostaglandin E2 Synthesis

alpha-Lipoic acid (LA) has been intensely investigated as a therapeutic agent for several pathological conditions, including diabetic polyneuropathy. In the present study, we examined the effects of LA on osteoclastic bone loss associated with inflammation. LA significantly inhibited IL-1-induced osteoclast formation in cocultures of mouse osteoblasts and bone marrow cells, but LA had only a marginal effect on osteoclastogenesis from bone marrow macrophages induced by receptor activator of NF-kappaB ligand (RANKL). LA inhibited both the sustained up-regulation of RANKL expression and the production of PGE2 induced by IL-1 in osteoblasts. In addition, treatment with either prostaglandin E2 (PGE2) or RANKL rescued IL-1-induced osteoclast formation inhibited by LA or NS398, a specific cyclooxygenase-2 (COX-2) inhibitor, in cocultures. LA blocked IL-1-induced PGE2 production even in the presence of arachidonic acid, without affecting the expression of COX-2 and membrane-bound PGE2 synthase. Dihydrolipoic acid (the reduced form of LA), but not LA, attenuated recombinant COX-2 activity in vitro. LA also inhibited osteoclast formation and bone loss induced by IL-1 and LPS in mice. Our results suggest that the reduced form of LA inhibits COX-2 activity, PGE2 production, and sustained RANKL expression, thereby inhibiting osteoclast formation and bone loss in inflammatory conditions.

Prostaglandin E2 Strongly Inhibits Human Osteoclast Formation

Prostaglandin E(2) (PGE(2)) enhances osteoclast formation in mouse macrophage cultures treated with receptor activator of nuclear factor-kappaB ligand (RANKL). The effects of PGE(2) on human osteoclast formation were examined in cultures of CD14(+) cells prepared from human peripheral blood mononuclear cells. CD14(+) cells differentiated into osteoclasts in the presence of RANKL and macrophage colony-stimulating factor. CD14(+) cells expressed EP2 and EP4, but not EP1 or EP3, whereas CD14(+) cell-derived osteoclasts expressed none of the PGE(2) receptors. PGE(2) and PGE(1) alcohol (an EP2/4 agonist) stimulated cAMP production in CD14(+) cells. In contrast to mouse macrophage cultures, PGE(2) and PGE(1) alcohol inhibited RANKL-induced human osteoclast formation in CD14(+) cell cultures. H-89 blocked the inhibitory effect of PGE(2) on human osteoclast formation. These results suggest that the inhibitory effect of PGE(2) on human osteoclast formation is mediated by EP2/EP4 signals. SaOS4/3 cells have been shown to support human osteoclast formation in cocultures with human peripheral blood mononuclear cells in response to PTH. PGE(2) inhibited PTH-induced osteoclast formation in cocultures of SaOS4/3 cells and CD14(+) cells. Conversely, NS398 (a cyclooxygenase 2 inhibitor) enhanced osteoclast formation induced by PTH in the cocultures. The conditioned medium of CD14(+) cells pretreated with PGE(2) inhibited RANKL-induced osteoclast formation not only in human CD14(+) cell cultures, but also in mouse macrophage cultures. These results suggest that PGE(2) inhibits human osteoclast formation through the production of an inhibitory factor(s) for osteoclastogenesis of osteoclast precursors.

Bacterial components and bone resorption

Osteoclasts are bone-resorbing multinucleated cells derived from the monocyte/macrophage lineage under the control of bone-forming osteoblasts. Bone-resorbing factors such as 1α,25(OH)2D3 and IL-1 stimulated osteoclast formation in cocultures of mouse osteoblasts and hemopoietic cells through the induction of RANKL expression in osteoblasts. We examined the mechanism of action of LPS and MDP in osteoclastic bone resorption using the coculture system. LPS stimulated osteoclast formation through the enhancement of RANKL expression in osteoblasts. LPS also stimulated the survival of mature osteoclasts. Using MyD88-deficient mice, we showed that MyD88 was involved in both RANKL expression in osteoblasts and the survival of osteoclasts induced by LPS. On the other hand, MDP alone could not induce osteoclast formation in the coculture, but enhanced it induced LPS but not 1α,25(OH)2D3. LPS stimulated Nod2 expression in osteoblasts, but 1α,25(OH)2D3 did not. MDP enhanced RANKL expression induced by LPS in osteoblasts. The depletion of intracellular Nod2 by siRNA blocked the MDP-induced upregulation of RANKL mRNA in osteoblasts. These results suggest that bacterial components stimulate osteoclastic bone resorption through several signaling pathways including TLR/MyD88- and Nod-mediated ones.

Muramyl Dipeptide Enhances Osteoclast Formation Induced by Lipopolysaccharide, IL-1α, and TNF-α through Nucleotide-Binding Oligomerization Domain 2-Mediated Signaling in Osteoblasts

Muramyl dipeptide (MDP) is the minimal essential structural unit responsible for the immunoadjuvant activity of peptidoglycan. As well as bone-resorbing factors such as 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) and PGE2, LPS and IL-1alpha stimulate osteoclast formation in mouse cocultures of primary osteoblasts and hemopoietic cells. MDP alone could not induce osteoclast formation in the coculture, but enhanced osteoclast formation induced by LPS, IL-1alpha, or TNF-alpha but not 1alpha,25(OH)2D3 or PGE2. MDP failed to enhance osteoclast formation from osteoclast progenitors induced by receptor activator of NF-kappaB ligand (RANKL) or TNF-alpha. MDP up-regulated RANKL expression in osteoblasts treated with LPS or TNF-alpha but not 1alpha,25(OH)2D3. Osteoblasts expressed mRNA of nucleotide-binding oligomerization domain 2 (Nod2), an intracellular sensor of MDP, in response to LPS, IL-1alpha, or TNF-alpha but not 1alpha,25(OH)2D3. Induction of Nod2 mRNA expression by LPS but not by TNF-alpha in osteoblasts was dependent on TLR4 and MyD88. MDP also enhanced TNF-alpha-induced osteoclast formation in cocultures prepared from Toll/IL-1R domain-containing adapter protein (TIRAP)-deficient mice through the up-regulation of RANKL mRNA expression in osteoblasts, suggesting that TLR2 is not involved in the MDP-induced osteoclast formation. The depletion of intracellular Nod2 by small interfering RNA blocked MDP-induced up-regulation of RANKL mRNA in osteoblasts. LPS and RANKL stimulated the survival of osteoclasts, and this effect was not enhanced by MDP. These results suggest that MDP synergistically enhances osteoclast formation induced by LPS, IL-1alpha, and TNF-alpha through RANKL expression in osteoblasts, and that Nod2-mediated signals are involved in the MDP-induced RANKL expression in osteoblasts.

Phosphodiesterase inhibitors stimulate osteoclast formation via TRANCE/RANKL expression in osteoblasts: possible involvement of ERK and p38 MAPK pathways

Phosphodiesterases (PDEs) are enzymes that degrade intracellular cAMP. In the present study, 3-isobutyl-1-methylxanthine (IBMX) and pentoxifylline, PDE inhibitors, induced osteoclast formation in cocultures of mouse bone marrow cells and calvarial osteoblasts. These inhibitors induced the expression of the osteoclast differentiation factor, TNF-related activation induced cytokine (TRANCE, identical to RANKL, ODF, and OPGL), in calvarial osteoblasts. IBMX induced phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) in osteoblasts. Induction of TRANCE expression by IBMX was partially suppressed by the inhibitors of protein kinase A (PKA), ERK, and p38 MAPK, suggesting that activation of ERK and p38 MAPK, as well as PKA, is involved in TRANCE expression by IBMX. Osteoblasts expressed PDE4, a PDE subtype, and rolipram, a selective inhibitor of PDE4, induced TRANCE expression. These results suggest that PDE4 is a key regulator of TRANCE expression in osteoblasts, which in turn controls osteoclast formation.

Rolipram, a Phosphodiesterase 4 inhibitor, stimulates osteoclast formation by inducing TRANCE expression in mouse calvarial cells

Phosphodiesterase (PDE) 4 is an enzyme that degrades intracellular cAMP. In the present study, the effect of rolipram, a specific phosphodiesterase (PDE) 4 inhibitor, on osteoclast formation was investigated. Rolipram induced osteoclast formation in cocultures of mouse bone marrow cells and calvarial osteoblasts. This activity was not observed in the absence of calvarial osteoblasts, suggesting that calvarial osteoblasts are likely target cells of rolipram. Osteoclast formation by rolipram was completely blocked by the addition of osteoprotegerin (OPG), a soluble decoy receptor for the osteoclast differentiation factor, TNF-related activation-induced cytokine (TRANCE, identical to RANKL, ODF, and OPGL). Northern blot analysis revealed the effect of rolipram to be associated with the increased expression of TRANCE mRNA in mouse calvarial osteoblasts. Collectively, these data indicate that PDE4 inhibitor up-regulates the TRANCE mRNA expression in osteoblasts, which in turn controls osteoclast formation.

Secreted frizzled-related protein-1 inhibits RANKL-dependent osteoclast formation.

We determined that sFRP-1 mRNA was differentially expressed by osteoblast/stromal cell lines and that sFRP-1 neutralizing antibodies and siRNA complementary to sFRP-1 coding sequence enhanced, while recombinant sFRP-1 inhibited, osteoclast formation. In studying the mechanism of action for sFRP-1, we found that sFRP-1 could bind recombinant RANKL. These results suggest potential cross-talk between Wnt and RANKL pathways. Osteoclast formation in normal bone remodeling requires the presence of osteoblast lineage cells that express RANKL and macrophage-colony-stimulating factor (M-CSF), which interact with their cognate receptors on the osteoclast precursor. We identified secreted Frizzled-related protein-1 (sFRP-1), which is known to bind to Wnt and inhibit the Wnt signaling pathway, as an osteoblast-derived factor that impinges on osteoclast formation and activity. Differential display of mRNA from osteoblast lineage cell lines established sFRP-1 to be highly expressed in an osteoclast supporting cell line. sFRP-1 expression in bone was determined by in situ hybridization, and the effects of sFRP-1 on osteoclast formation were determined using a neutralizing antibody, siRNA, for sFRP-1 and recombinant protein. In situ hybridization revealed sFRP-1 mRNA expression in osteoblasts and chondrocytes in murine bone. sFRP-1 mRNA expression could be elevated in calvarial primary osteoblasts in response to prostaglandin E2 (PGE2) or interleukin (IL)-11, whereas many other osteotropic agents (e.g., IL-1, IL-6, calcitrol, parathyroid hormone) were without any effect. In vitro assays of osteoclast formation established sFRP-1 to be an inhibitor of osteoclast formation. Neutralizing antibodies against sFRP-1 enhanced TRACP+ mononuclear and multinuclear osteoclast formation (3- and 2-fold, respectively) in co-cultures of murine osteoblasts with spleen cells, whereas siRNA complementary to sFRP-1 coding sequence significantly enhanced osteoclast formation in co-cultures of KUSA O (osteoblast/stromal cell line) and bone marrow cells, cultured in the presence of PGE2 and 1,25(OH)2 vitamin D3. Recombinant sFRP-1 dose-dependently inhibited osteoclast formation in osteoblast/spleen co-cultures, RANKL + M-CSF-treated splenic cultures, and RANKL-treated RAW264.7 cell cultures, indicating a direct action of sFRP-1 on hematopoietic cells. Consistent with this, sFRP-1 was found to bind to RANKL in ELISAs. sFRP-1 is expressed by osteoblasts and inhibits osteoclast formation. While sFRP-1 activity might involve the blocking of endogenous Wnt signaling, our results suggest that, alternatively, it could be because of direct binding to RANKL. This study describes a new mechanism whereby osteoblasts regulate osteoclastogenesis through the expression and release of sFRP-1.

Induction of RANKL expression and osteoclast maturation by the binding of fibroblast growth factor 2 to heparan sulfate proteoglycan on rheumatoid synovial fibroblasts.

Rheumatoid arthritis (RA) is characterized by progressive joint destruction. The aim of this study was to clarify the relevance of RA synovial fibroblasts (RASFs) and fibroblast growth factor 2 (FGF-2), which is produced abundantly by RASFs, to the osteoclastogenesis and bone resorption in RA. Synovial fibroblasts were prepared from the synovial tissues of 10 patients with active RA and 7 patients with osteoarthritis (OA). The expression of RANKL, intercellular adhesion molecule 1 (ICAM-1), FGF receptor 1 (FGFR-1), and heparan sulfate proteoglycan (HSPG) on synovial fibroblasts was measured by FACScan. Osteoclast formation in cocultures of RASFs and peripheral blood mononuclear cells (PBMCs) was evaluated by tartrate-resistant acid phosphatase staining and a pit-formation assay using dentin slices. FGF-2 induced the expression of both RANKL and ICAM-1 on RASFs more so than on OA synovial fibroblasts (OASFs). FGF-2-induced up-regulation of RANKL and ICAM-1 was inhibited by anti-FGF-2 antibody. Although FGFR-1 was equally expressed on RASFs and OASFs, HSPG was highly expressed on RASFs. Up-regulation of RANKL by FGF-2 on RASFs was diminished by the removal of heparan sulfate with heparitinase. Osteoclast formation from PBMCs induced by RASFs was inhibited by the addition of either heparitinase, anti-ICAM-1 antibody, anti-FGF-2 antibody, or osteoprotegerin. FGF-2-induced RANKL on RASFs and osteoclast formation were suppressed by an inhibitor of ERK. FGF-2 was transferred to FGFR-1 through binding to HSPG, which is characteristically expressed on RASFs, resulting in RANKL- and ICAM-1-mediated maturation of osteoclasts via ERK activation. Thus, we propose that FGF-2 not only augments the proliferation of RASFs, but also is involved in osteoclast maturation, which leads to bone destruction in RA.

Macrophage inflammatory protein-1alpha induces hypercalcemia in adult T-cell leukemia.

Hypercalcemia is observed in >80% of ATL. Serum MIP-1alpha levels were elevated in all 24 ATL with hypercalcemia but undetectable in all 10 patients with humoral hypercalcemia of malignancy with solid tumors and in 34 of 37 ATL without hypercalcemia. We propose that serum MIP-1alpha is a clinical hallmark for hypercalcemia in ATL. High serum cytokines levels are not always associated with hypercalcemia in patients with adult T-cell leukemia (ATL), suggesting that other factors are involved in the pathogenesis of ATL patients with hypercalcemia. This study was designed to determine the role of macrophage inflammatory protein-1alpha (MIP-1alpha), a chemokine recently described as an osteoclast stimulatory factor, in ATL-associated hypercalcemia. We measured serum interleukin (IL)-1beta, IL-6, TNF-alpha, parathyroid hormone-related protein (PTHrP), and MIP-1alpha levels in ATL patients by enzyme-linked immunosorbent assays. FACScan was used to measure the expression of RANKL on ATL cells. Osteoclast formation in cocultures of ATL cells and peripheral blood mononuclear cells (PBMCs) was evaluated by TRACP staining. High serum MIP-1alpha levels were noted in all 24 ATL patients with hypercalcemia and in 3 of 37 ATL patients without hypercalcemia. The elevated levels of MIP-1alpha and calcium in ATL patients decreased after effective chemotherapy, emphasizing the role of MIP-1alpha in ATL hypercalcemia. ATL cells spontaneously produced MIP-1alpha. MIP-1alpha significantly enhanced human monocyte (precursor cells of osteoclasts) migration and induced RANKL expression on ATL cells. ATL cell-induced osteoclast formation from PBMCs was inhibited by anti-MIP-1alpha antibody and osteoprotegerin. Our results suggest that MIP-1alpha can induce RANKL on ATL cells in autocrine fashion and that RANKL seems to mediate the hypercalcemic effect of MIP-1alpha in ATL. We propose that MIP-1alpha is the clinical hallmark of hypercalcemia in ATL and could be a potentially useful therapeutic target.

Suppression of Osteoprotegerin Expression by Prostaglandin E2 Is Crucially Involved in Lipopolysaccharide-Induced Osteoclast Formation

LPS is a potent stimulator of bone resorption in inflammatory diseases. The mechanism by which LPS induces osteoclastogenesis was studied in cocultures of mouse osteoblasts and bone marrow cells. LPS stimulated osteoclast formation and PGE(2) production in cocultures of mouse osteoblasts and bone marrow cells, and the stimulation was completely inhibited by NS398, a cyclooxygenase-2 inhibitor. Osteoblasts, but not bone marrow cells, produced PGE(2) in response to LPS. LPS-induced osteoclast formation was also inhibited by osteoprotegerin (OPG), a decoy receptor of receptor activator of NF-kappaB ligand (RANKL), but not by anti-mouse TNFR1 Ab or IL-1 receptor antagonist. LPS induced both stimulation of RANKL mRNA expression and inhibition of OPG mRNA expression in osteoblasts. NS398 blocked LPS-induced down-regulation of OPG mRNA expression, but not LPS-induced up-regulation of RANKL mRNA expression, suggesting that down-regulation of OPG expression by PGE(2) is involved in LPS-induced osteoclast formation in the cocultures. NS398 failed to inhibit LPS-induced osteoclastogenesis in cocultures containing OPG knockout mouse-derived osteoblasts. IL-1 also stimulated PGE(2) production in osteoblasts and osteoclast formation in the cocultures, and the stimulation was inhibited by NS398. As seen with LPS, NS398 failed to inhibit IL-1-induced osteoclast formation in cocultures with OPG-deficient osteoblasts. These results suggest that IL-1 as well as LPS stimulates osteoclastogenesis through two parallel events: direct enhancement of RANKL expression and suppression of OPG expression, which is mediated by PGE(2) production.

The effect of deletion of cyclooxygenase-2, prostaglandin receptor EP2, or EP4 in bone marrow cells on osteoclasts induced by mouse mammary cancer cell lines

The inducible prostaglandin (PG) synthesis enzyme, cyclooxygenase-2 (COX-2), is involved in osteoclast (OC) formation in cocultures of mouse mammary cancer cell lines (MMT060562 or BALB/c-MC) and bone marrow cells through production of PGE 2. There are four PGE 2 receptors but only the EP2 and EP4 receptors are reported to be important for OC formation. We have investigated the role of COX-2, EP2 receptor, and EP4 receptor in marrow cells for osteoclastogenesis in cocultures of cancer cells and bone marrow cells. We cocultured cancer cell lines with bone marrow cells from COX-2 knockout (−/−), EP2 −/− or EP4 −/− mice compared to wild-type mice. In addition, an EP4 receptor antagonist (EP4 RA) was added in some cocultures. Disruption of COX-2 gene in bone marrow cells had no effect on PGE 2 production and OC formation in cocultures with MMT060562, while it abrogated PGE 2 production and OC formation in cocultures with BALB/c-MC. Disruption of the EP2 gene in bone marrow cells had no effect on OC formation in the cocultures, while disruption of the EP4 gene in bone marrow cells abrogated OC formation in the cocultures. Furthermore, EP4 RA suppressed OC formation and prevented the increase in receptor activator of nuclear factor κB ligand (RANKL) mRNA levels in the cocultures. We conclude that COX-2 in cancer cells is responsible for PGE 2 and OC production in cocultures with MMT060562, while COX-2 in bone marrow cells, not cancer cells, is responsible for PGE 2 and OC production in cocultures with BALB/c-MC, and EP4 receptors are essential for OC formation in both cocultures.

Inhibition of osteoclastogenesis by a phosphodiesterase 4 inhibitor XT-611 through synergistic action with endogenous prostaglandin E 2

We examined the effect of a phosphodiesterase 4 (PDE4) inhibitor, 3,4-dipropyl-4,5,7,8-tetrahydro-3 H-imidazo[1,2- i]-purin-5-one (XT-611) on osteoclast formation in three different mouse bone-marrow cell (BMC) culture systems. We confirmed that selective inhibitors of PDE4, including XT-611, among several PDE inhibitors decreased osteoclast formation in the BMC culture system. XT-611 also inhibited osteoclast formation in co-culture of mouse bone-marrow stromal cell line ST2 and adherent cell-depleted (ACD)-BMCs. However, it did not inhibit osteoclastogenesis in culture of ACD-BMCs alone in the presence of macrophage-colony stimulating factor (M-CSF) and soluble receptor activator of NF-κB ligand (sRANKL). XT-611 significantly increased prostaglandin E 2 (PGE 2) production from ST2 cells and, in combination with PGE 2, synergistically increased cAMP concentration in osteoclast progenitors. In the ST2 co-culture system, XT-611 did not influence the expression of RANKL, osteoprotegerin and RANK mRNAs. By combined treatment with XT-611 and PGE 2 of ACD-BMCs, osteoclast multinucleation was clearly inhibited with decrease in the expression of calcitonin receptor mRNA, while the expression of RANK and c-fms (an M-CSF receptor) mRNAs was unchanged. These results indicate that the PDE4 inhibitor inhibits osteoclastogenesis by acting on osteoclast progenitors synergistically with PGE 2 secreted from stromal cells, but not by influencing the cell-to-cell interaction between stromal cells and osteoclast progenitors.

RANK-Fc: a therapeutic antagonist for RANK-L in myeloma.

Severe bone destruction due to inappropriate osteoclastogenesis is a prominent feature of multiple myeloma (MM). MM increases bone loss by disrupting the checks that normally control signaling by receptor activator of nuclear factor kappaB ligand (RANK-L, also called TRANCE [tumor necrosis factor-related, activation-induced cytokine], osteoprotegerin ligand [OPG-L], osteoclast differentiation factor [ODF], and tumor necrosis factor superfamily member 11 [TNFSF11]), a TNF-family cytokine required for osteoclast differentiation and activation. RANK-L binds to its functional receptor RANK (TNF receptor superfamily member 11a [TNF RSF11a]) to stimulate osteoclastogenesis. Osteotropic cytokines regulate this process by controlling bone marrow stromal expression of RANK-L. Further control over osteoclastogenesis is maintained by regulated expression of osteoprotegerin (OPG, also called osteoclastogenesis inhibitory factor and TNFRSF11b), a soluble decoy receptor for RANK-L. In normal bone marrow, abundant stores of OPG in stroma, megakaryocytes, and myeloid cells provide a natural buffer against increased RANK-L. MM disrupts these controls by increasing expression of RANK-L and decreasing expression of OPG. Concurrent deregulation of RANK-L and OPG expression is found in bone marrow biopsies from patients with MM but not in specimens from patients with non-MM hematologic malignancies. RANK-Fc is a recombinant RANK-L antagonist that is formed by fusing the extracellular domain of RANK to the Fc portion of human immunoglobulin G(1) (hIgG(1)). In vitro, addition of RANK-Fc virtually eliminates the formation of osteoclasts in cocultures of MM with bone marrow and osteoblast/stromal cells. The severe combined immunodeficiency (SCID)/ARH77 mouse model and the SCID-hu-MM mouse model of human MM were used to assess the ability of RANK-Fc to block the development of MM-induced bone disease in vivo. Mice received either RANK-Fc or hIgG(1) 200 microg intravenously three times per week. RANK-Fc limited bone destruction in both the SCID/ARH-77 model and the SCID-hu-MM model. Administration of RANK-Fc also caused a marked reduction in tumor burden and serum paraprotein in SCID-hu-MM mice that was associated with the restoration of OPG and a reduction in RANK-L expression in the xenograft. MM-induced bone destruction requires increased RANK-L expression and is facilitated by a concurrent reduction in OPG, a natural decoy receptor for RANK-L. Administration of the RANK-L antagonist RANK-Fc limits MM-induced osteoclastogenesis, development of bone disease, and MM tumor progression.

Involvement of cyclo-oxygenase-2 in osteoclast formation and bone destruction in bone metastasis of mammary carcinoma cell lines.

We previously reported that mouse mammary carcinoma cell lines (MMT060562 and BALB/c-MC) induced osteoclast formation through production of prostaglandin E2 (PGE2) in cocultures with mouse bone marrow cells, but the mechanism(s) of PG production remained unclear. In the present in vitro and in vivo studies, we tested the involvement of cyclo-oxygenase-2 (COX-2), an inducible rate-limiting enzyme in PG biosynthesis, in the stimulation of osteoclast formation by mouse mammary carcinoma cell lines. Addition of a selective COX-2 inhibitor, JTE-522, to cocultures of mammary carcinoma cell lines and bone marrow cells lowered PGE2 concentration in the culture media and inhibited osteoclast formation in a dose-dependent manner. Northern blotting showed a very high level of COX-2 messenger RNA (mRNA) expression in MMT060562. The mRNA expression was low in BALB/c-MC, but it increased when BALB/c-MC and bone marrow cells were cocultured. The results of immunocytochemistry for COX-2 protein in respective cultures were compatible with the results of COX-2 mRNA. In vivo, BALB/c-MC injected into the heart of Balb/c mice metastasized to bone and formed osteolytic lesions in their hindlimbs. Histological examination revealed that tumor cells had metastasized to the bone marrow cavity and destroyed the bone trabeculae. Immunohistochemistry demonstrated that bone marrow stromal cells adjacent to tumor cells expressed COX-2 protein. These findings suggest that COX-2 plays an important role in the osteolysis of bone metastasis in vivo as well as in osteoclast formation in cocultures used as an in vitro model of metastatic bone disease.

Breast Cancer Increases Osteoclastogenesis by Secreting M-CSF and Upregulating RANKL in Stromal Cells

Background. Breast cancer metastasis to bone causes resorption of the mineralized matrix by osteoclasts.Macrophage colony stimulating factor (M-CSF)and receptor activator of the NF-κB ligand (RANKL) are produced by stromal cells and are essential for osteoclast formation. The human breast cancer cell line, MDA-MB-231, reliably forms bone metastases in a murine model and stimulates osteoclast formation in culture. We hypothesized that MDA-MB-231 stimulates osteoclast formation through secretion of M-CSF and/or RANKL.Materials and methods. We cocultured MDA-MB-231 and a bone marrow derived cell line, UAMS-33, and evaluated the expression of M-CSF and RANKL mRNA. Osteoclast formation was assessed using these cells added to hematopoietic cell cultures.Results. MDA-MB-231 exhibited constitutive expression of M-CSF mRNA. As expected, addition of recombinant M-CSF (30 ng/ml) and RANKL (30 ng/ml) to hematopoietic osteoclast precursors supported osteoclast formation, while the addition of soluble RANKL alone or MDA-231 without added RANKL did not. Notably, coculture of MDA-231 with hematopoietic cells and added soluble RANKL stimulated significant osteoclast formation, indicating that MDA-231 served as an effective source for M-CSF. MDA-231 did not express RANKL. However, when cocultured with the murine bone marrow stromal cell line UAMS-33, RANKL expression was significantly increased in the latter cells. MDA-231 also stimulated osteoclast formation in coculture with UAMS-33 and hematopoietic cells.Conclusions. We conclude that MDA-MB-231 increases osteoclast formation by secreting adequate amounts of M-CSF protein and enhancing the expression of RANKL by stromal support cells. The ability to stimulate osteoclasts may explain the ability to metastasize to bone.

Possible involvement of protein kinases and Smad2 signaling pathways on osteoclast differentiation enhanced by activin A.

Bone tissues reportedly contain considerable amounts of activin A and follistatin, an activin A-binding protein. In the present study, we found that follistatin strongly inhibited osteoclast formation in cocultures of mouse bone marrow cells and primary osteoblasts induced by 1alpha,25 dihydroxyvitamin D(3), prostaglandin E(2), and interleukin-1alpha. Antibody aganist activin A also inhibited the osteoclast formation. Furthermore, activin A synergistically stimulated osteoclast differentiation mediated by receptor activator NF-kappaB ligand (RANKL). RT-PCR analysis revealed that osteoblasts produced not only activin A but also follistatin. Western blot analysis of a panel of phosphorylated proteins revealed that activin A stimulated the phosphorylation of p44/42 mitogen activated protein (MAP) kinase (ERK1/2) and p38 MAP kinase in macrophage colony-stimulating factor-dependent bone marrow macrophages (M-BMMPhis). In addition, phosphorylation of Smad2 was observed in M-BMMPhis stimulated with activin A. These findings indicate that the phosphorylation of p44/42 MAP kinase, p38 MAP kinase, and Smad2 is involved in activin A-enhanced osteoclast differentiation induced by RANKL. Taken together, these results suggest that both activin A and follistatin produced by osteoblasts may play an important role in osteoclast differentiation through MAP kinases and Smad2 signaling pathways.

IL-12 alone and in synergy with IL-18 inhibits osteoclast formation in vitro.

IL-12, like IL-18, was shown to potently inhibit osteoclast formation in cultures of cocultures of murine osteoblast and spleen cells, as well as in adult spleen cells treated with M-CSF and receptor activator of NF-kappaB ligand (RANKL). Neither IL-12 nor IL-18 was able to inhibit RANKL-induced osteoclast formation in cultured RAW264.7 cells, demonstrating that IL-12, like IL-18, was unable to act directly on osteoclastic precursors. IL-12, like IL-18, was found to act by T cells, since depletion of T cells from the adult spleen cell cultures ablated the inhibitory action of IL-12 and addition of either CD4 or CD8 T cells from C57BL/6 mice to RANKL-stimulated RAW264.7 cultures permitted IL-12 or IL-18 to be inhibitory. Additionally, IL-12 was still able to inhibit osteoclast formation in cocultures with osteoblasts and spleen cells from either GM-CSF R(-/-) mice or IFN-gamma R(-/-) mice, indicating that neither GM-CSF nor IFN-gamma was mediating osteoclast inhibition in these cultures. Combined, IL-18 and IL-12 synergistically inhibited osteoclast formation at concentrations 20- to 1000-fold less, respectively, than when added individually. A candidate inhibitor could not be demonstrated using neutralizing Abs to IL-4, IL-10, or IL-13 or from mRNA expression profiles among known cytokine inhibitors of osteoclastogenesis in response to IL-12 and IL-18 treatment, although the unknown inhibitory molecule was determined to be secreted from T cells.

Intracellular calcium and protein kinase C mediate expression of receptor activator of nuclear factor-kappaB ligand and osteoprotegerin in osteoblasts.

Receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) produced by osteoblasts/stromal cells are involved as positive and negative regulators in osteoclast formation. Three independent signals have been proposed to induce RANKL expression in osteoblasts/stromal cells: vitamin D receptor-, cAMP-, and gp130-mediated signals. We previously reported that intracellular calcium-elevating compounds such as ionomycin, cyclopiazonic acid, and thapsigargin induced osteoclast formation in cocultures of mouse bone marrow cells and primary osteoblasts. Increases in calcium concentration in culture medium also induced osteoclast formation in cocultures. Treatment of primary osteoblasts with these compounds or with high calcium medium stimulated the expression of both RANKL and OPG messenger RNAs (mRNAs). 1,2-Bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)-tetra(acetoxymethyl)ester, an intracellular calcium chelator, suppressed both ionomycin-induced osteoclast formation in cocultures and expression of RANKL and OPG mRNAs in primary osteoblasts. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, also stimulated osteoclast formation in these cocultures and the expression of RANKL and OPG mRNAs in primary osteoblasts. Protein kinase C inhibitors such as calphostin and staurosporin suppressed ionomycin- and PMA-induced osteoclast formation in cocultures and expression of RANKL and OPG mRNAs in primary osteoblasts. Ionomycin stimulated RANKL mRNA expression in ST2 and MC3T3-G2/PA6 cells, but not in MC3T3-E1 or NIH-3T3 cells. These effects were closely correlated with osteoclast formation in response to ionomycin in cocultures with these stromal cell lines. OPG strongly inhibited osteoclast formation induced by calcium-elevating compounds and PMA in cocultures, suggesting that RANKL expression in osteoblasts is a rate-limiting step for osteoclast induction. Forskolin, an activator of cAMP signals, also stimulated osteoclast formation in cocultures. Forskolin enhanced RANKL mRNA expression but suppressed OPG mRNA expression in primary osteoblasts. These results suggest that the calcium/protein kinase C signal in osteoblasts/stromal cells is the fourth signal for inducing RANKL mRNA expression, which, in turn, stimulates osteoclast formation.

Microarchitectural assessment of bone

Basic fibroblast growth factor (bFGF) induced osteoclast formation in co-cultures of mouse spleen cells and osteoblasts. Osteoclastogenesis inhibitory factor (OCIF) and a selective cyclooxygenase-2 (COX-2) inhibitor, NS-398, abolished bFGF-induced osteoclast formation. bFGF did not affect spleen cells, but it did affect osteoblasts, to stimulate osteoclast formation. Northern blot analysis revealed that bFGF up-regulated the expression of osteoclast differentiation factor (ODF) and COX-2 and down-regulated the expression of OCIF in primary osteoblastic cells. NS-398 abolished the increase of ODF mRNA, but it had no effect on the decrease of OCIF mRNA. NS-398 suppressed the binding of 125I-labeled OCIF to osteoblastic cells treated with bFGF. Enzyme-linked immunosorbent assay showed that bFGF inhibited OCIF production by osteoblastic cells, and the inhibition was not affected by NS-398. We conclude that bFGF induces osteoclast formation by stimulating ODF production through COX-2-mediated prostaglandin synthesis and by suppressing OCIF production through a mechanism independent of prostaglandin synthesis.

Low Calcium Environment Effects Osteoprotegerin Ligand/Osteoclast Differentiation Factor

In coculture with osteoblastic cell line MC3T3-E1 (E1) and mouse bone marrow cells, we reported that numbers of osteoclasts rose significantly on exposure to a low-calcium environment. Here we examined how osteoblasts influence osteoclastogenesis under a low-calcium environment. Comparing low extracellular calcium with a regular calcium environment, osteoprotegerin ligand (OPGL)/osteoclast differentiation factor (ODF) mRNA expression show more increase in the culture of low-calcium environment than in that of a regular calcium environment. Calcium-sensing receptor (CaSR), which was supposed as one of the mechanisms of recognizing extracellular calcium, existedon the surface of E1 cells. When E1 cells stimulated with agonists of CaSR, gadolinium, and neomycin, OPGL/ODF mRNA expression decreased. Moreover, these agonists reduced osteoclast formation in coculture. Taken together, it is possible that osteoblasts may recognize extracellular calcium via CaSR and regulate osteoclastogenesis.