Vitamin D antagonist, TEI-9647, inhibits osteoclast formation induced by 1alpha,25-dihydroxyvitamin D3 from pagetic bone marrow cells

(23S)-25-Dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) functions an antagonist of the 1alpha,25-dihydroxyvitamin D(3) (1alpha,25-(OH)(2)D(3)) nuclear receptor (VDR)-mediated differentiation of human leukemia (HL-60) cells [J. Biol. Chem. 274 (1999) 16392]. We examined the effect of vitamin D antagonist, TEI-9647, on osteoclast formation induced by 1alpha,25-(OH)(2)D(3) from bone marrow cells of patients with Paget's disease. TEI-9647 itself never induced osteoclast formation even at 10(-6)M, but dose-dependently (10(-10) to 10(-6)M) inhibited osteoclast formation induced by physiologic concentrations of 1alpha,25-(OH)(2)D(3) (41 pg/ml, 10(-10)M) from bone marrow cells of patients with Paget's disease. At the same time, 10(-8)M of TEI-9647 alone did not cause 1alpha,25-(OH)(2)D(3) dependent gene expression, but almost completely suppressed TAF(II)-17, a potential coactivator of VDR and 25-hydroxyvitamin D(3)-24-hydroxylase (25-OH-D(3)-24-hydroxylase) gene expression induced by 10(-10)M 1alpha,25-(OH)(2)D(3) in bone marrow cells of patients with Paget's disease. Moreover, TEI-9647 dose-dependently inhibited bone resorption induced by 10(-9)M 1alpha,25-(OH)(2)D(3) by osteoclasts produced by RANKL and M-CSF treatment of measles virus nucleocapsid gene transduced bone marrow cells. These results suggest that TEI-9647 acts directly on osteoclast precursors and osteoclasts, and that TEI-9647 may be a novel agent to suppress the excessive bone resorption and osteoclast formation in patients with Paget's disease.     

Muramyl dipeptide enhances osteoclast formation induced by lipopolysaccharide, IL-1 alpha, and TNF-alpha 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.     

Lack of bone resorption in osteosclerotic (oc/oc) mice is due to a defect in osteoclast progenitors rather than the local microenvironment provided by osteoblastic cells.

In a co-culture system of mouse spleen cells and osteoblastic cells, we have demonstrated that a suitable microenvironment must be provided by osteoblastic cells in order for osteoclast-like multinucleated cell (MNC) formation. Using this co-culture system, we examined the pathogenetic mechanism underlying the lack of bone resorption in osteosclerotic oc/oc mice. Numerous tartrate-resistant acid phosphatase (TRAP, an osteoclast marker enzyme)-positive MNCs were formed in response to 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] both in co-cultures of oc/oc spleen cells and normal osteoblastic cells and in those of normal spleen cells and oc/oc osteoblastic cells. TRAP-positive MNCs derived from normal spleen cells tended to spread out on culture dishes, whereas those from oc/oc spleen cells remained as small, compact MNCs. When TRAP-positive MNCs enriched from co-cultures of normal spleen cells and oc/oc osteoblastic cells were cultured on dentine slices, they formed numerous resorption pits with ruffled borders and clear zones. In contrast, none of the TRAP-positive MNCs derived from oc/oc spleen cells formed either ruffled borders or resorption pits. These results indicate that the lack of bone resorption in oc/oc mice is due to a defect in osteoclast progenitors rather than the local microenvironment provided by osteoblastic cells.     

Vitamin D and bone

It is now well established that supraphysiological doses of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] stimulate bone resorption. Recent studies have established that osteoblasts/stromal cells express receptor activator of NF-kappaB ligand (RANKL) in response to several bone-resorbing factors including 1alpha,25(OH)(2)D(3) to support osteoclast differentiation from their precursors. Osteoclast precursors which express receptor activator of NF-kappaB (RANK) recognize RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into osteoclasts in the presence of macrophage-colony stimulating factor (M-CSF). Osteoprotegerin (OPG) acts as a decoy receptor for RANKL. We also found that daily oral administration of 1alpha,25(OH)(2)D(3) for 14 days to normocalcemic thyroparathyroidectomized (TPTX) rats constantly infused with parathyroid hormone (PTH) inhibited the PTH-induced expression of RANKL and cathepsin K mRNA in bone. The inhibitory effect of 1alpha,25(OH)(2)D(3) on the PTH-induced expression of RANKL mRNA occurred only with physiological doses of the vitamin. Supraphysiological doses of 1alpha,25(OH)(2)D(3) increased serum Ca and expression of RANKL in vivo in the presence of PTH. These results suggest that the bone-resorbing activity of vitamin D does not occur at physiological dose levels in vivo. A certain range of physiological doses of 1alpha,25(OH)(2)D(3) rather suppress the PTH-induced bone resorption in vivo, supporting the concept that 1alpha,25(OH)(2)D(3) or its derivatives are useful for the treatment of various metabolic bone diseases such as osteoporosis and secondary hyperparathyroidism.     

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.     

Osteoblastic regulation of osteoclast survival: effect of calcitriol

Throughout life, bone is remodelled in a dynamic process which results in a balance between bone formation by osteoblasts and bone resorption by osteoclasts. It is now clearly established that osteoblasts/stromal cells are crucial for differentiation of osteoclasts, through a mechanism involving cell-to-cell contact. However, the possible involvement of osteoblasts and stromal cells in the survival of osteoclasts has not yet been clearly demonstrated. In this study, we assessed the influence of cellular microenvironment, especially osteoblasts, on the osteoclast survival. Our results have shown significant differences in osteoclastic survival between unfractionated bone cells and pure osteoclasts. Furthermore, we have shown that addition of 1.25(OH)2D3 to unfractionated bone cells resulted in a dose-dependent increase in osteoclast survival. Finally, we have shown that a conditioned medium obtained from rat osteoblastic cells cultured with calcitriol was able to increase significantly survival of pure osteoclasts. Taken together, these results strongly suggest that osteoblastic cells present in the bone microenvironment might play a role in the osteoclastic survival by producing soluble factor which modulate osteoclast apoptosis.     

Tumor necrosis factor-alpha induces differentiation of and bone resorption by osteoclasts

Osteoclast progenitors differentiate into mature osteoclasts in the presence of receptor activator of NF-kappaB (RANK) ligand on stromal or osteoblastic cells and monocyte macrophage colony-stimulating factor (M-CSF). The soluble RANK ligand induces the same differentiation in vitro without stromal cells. Tumor necrosis factor-alpha (TNF-alpha), a potent cytokine involved in the regulation of osteoclast activity, promotes bone resorption via a primary effect on osteoblasts; however, it remains unclear whether TNF-alpha can also directly induce the differentiation of osteoclast progenitors into mature osteoclasts. This study revealed that TNF-alpha directly induced the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), which produced resorption pits on bone in vitro in the presence of M-CSF. The bone resorption activity of TNF-alpha-induced MNCs was lower than that of soluble RANK ligand-induced MNCs; however, interleukin-1beta stimulated this activity of TNF-alpha-induced MNCs without an increase in the number of MNCs. In this case, interleukin-1beta did not induce TRAP-positive MNC formation. The osteoclast progenitors expressed TNF receptors, p55 and p75; and the induction of TRAP-positive MNCs by TNF-alpha was inhibited completely by an anti-p55 antibody and partially by an anti-p75 antibody. Our findings presented here are the first to indicate that TNF-alpha is a crucial differentiation factor for osteoclasts. Our results suggest that TNF-alpha and M-CSF play an important role in local osteolysis in chronic inflammatory diseases.     

1 alpha, 25-Dihydroxyvitamin D3 augments clonal growth of macrophages from rat bone marrow progenitor cells and modulates their function

1 alpha, 25-Dihydroxyvitamin D3 (1,25(OH)2D3) was shown to enhance (approximately 2 fold) the colony-stimulating factor-dependent clonal growth of macrophage colonies and clusters from rat bone marrow progenitor cells. The proliferative capacity of macrophage progenitors in liquid cultures was likewise augmented (2-3 fold). Mononuclear phagocytes (macrophages, for simplicity) developing in the presence of 1,25(OH)2D3 showed a reduced capacity of migration. 1,25(OH)2D3 administered at bone marrow culture initiation led to augmentation of the phagocytic capability of macrophages in four-day cultures and to its suppression in macrophages in seven-day cultures. The observed patterns of modulation of differentiation and function by 1,25(OH)2D3 differ from the patterns we found for mouse bone marrow cells. The results suggest that the differential response to hormones observed in different species may include responses to 1,25(OH)2D3.     

Basic fibroblast growth factor inhibits osteoclast-like cell formation

Basic fibroblast growth factor (bFGF) inhibited osteoclast-like cell formation in co-cultures of mouse bone marrow cells either with the mouse stromal cell line, ST2, or with primary osteoblastic cells. Basic FGF significantly inhibited the osteoclast-like cell formation, induced by 1α,25-dihydroxyvitamin D₃[1α, 25(OH)₂D₃] when the cytokine was added to the culture, at an intermediate stage, suggesting that bFGF inhibits the differentiation of the osteoclast progenitors. With regard to target cells, bFGF directly affected ST2; it increased [³H]thymidine uptake and decreased the number of alkaline phosphatase-positive cells. In contrast, bFGF had no inhibitory effect on the colony formation of bone marrow cells induced by macrophage colony stimulating factor in methylcellulose culture. In addition, ST2 cells treated with bFGF produced similar amounts of colony forming activity to those without the cytokine. These findings indicated that the bFGF is not involved in the proliferation of progenitor cells even in the presence of ST2 cells. Furthermore, bFGF inhibited osteoclast-like cell formation induced not only by 1α,25(OH)₂D₃, but also by prostaglandin E₂ and by interleukin-11. These results suggest that bFGF inhibits the common site of osteoclast-like cell formation, as induced by different mechanisms. Our data also indicated that the target cells for bFGF in inhibiting osteoclast formation are not osteoclast progenitors but stromal cells such as ST2 and osteoblastic cells, which support osteoclast development. © 1996 Wiley-Liss, Inc.     

TGF-beta 1 and IFN-gamma direct macrophage activation by TNF-alpha to osteoclastic or cytocidal phenotype

TNF-related activation-induced cytokine (TRANCE; also called receptor activator of NF-kappaB ligand (RANKL), osteoclast differentiation factor (ODF), osteoprotegerin ligand (OPGL), and TNFSF11) induces the differentiation of progenitors of the mononuclear phagocyte lineage into osteoclasts in the presence of M-CSF. Surprisingly, in view of its potent ability to induce inflammation and activate macrophage cytocidal function, TNF-alpha has also been found to induce osteoclast-like cells in vitro under similar conditions. This raises questions concerning both the nature of osteoclasts and the mechanism of lineage choice in mononuclear phagocytes. We found that, as with TRANCE, the macrophage deactivator TGF-beta(1) strongly promoted TNF-alpha-induced osteoclast-like cell formation from immature bone marrow macrophages. This was abolished by IFN-gamma. However, TRANCE did not share the ability of TNF-alpha to activate NO production or heighten respiratory burst potential by macrophages, or induce inflammation on s.c. injection into mice. This suggests that TGF-beta(1) promotes osteoclast formation not only by inhibiting cytocidal behavior, but also by actively directing TNF-alpha activation of precursors toward osteoclasts. The osteoclast appears to be an equivalent, alternative destiny for precursors to that of cytocidal macrophage, and may represent an activated variant of scavenger macrophage.     

1 alpha, 25-Dihydroxyvitamin D3 and analogues of vitamin D3 induce alkaline phosphatase activity in osteoblastic cells derived from newborn mouse calvaria

In order to study the effects of vitamin D metabolites on bone metabolism, clone MC3T3-E1 cells, which have retained osteoblastic activity, were cultured with various concentrations of the hormone, 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25 (OH)2D3]. A physiological concentration of 1 alpha, 25 (OH)2D3 stimulated alkaline phosphatase (ALP) activity in the cells. Other metabolites--1 alpha, 24-dihydroxyvitamin D3 [1 alpha, 24 (OH)2D3], 1 alpha-hydroxyvitamin D3 [1 alpha (OH)D3], and 24R,25-dihydroxyvitamin D3 [24R,25 (OH)2D3]--also induced increases in ALP activity in a dose-dependent fashion. However, their effective concentrations were 100 or 1,000 times greater than that of 1 alpha, 25 (OH)2D3. Hormone-induced and native ALP activities in the cells were of the same type as that found in newborn mouse calvaria; that is, they were heat-labile, L-homoarginine- and levamisole-sensitive, and L-phenylalanine-insensitive (liver-bone-kidney type). These results show that vitamin D metabolites stimulate bone formation in vitro and that they may be involved in bone formation in vivo as well.     

Osteoclast formation from mononuclear phagocytes: role of bone-forming cells

In a previous study, using co-cultures of embryonic bone rudiments stripped of periosteum, and mononuclear phagocytes of various sources, we found that multinucleated mineral-resorbing osteoclasts developed in vitro from radiosensitive mouse bone marrow mononuclear phagocytes (BMMP). (Burger, E. H., J. W. M. van der Meer, J. S. van de Gevel, C. W. Thesingh, and R. van Furth, 1982, J. Exp. Med. 156:1604-1614). In the present study, this co-culture technique was used to analyze the influence of bone-forming cells on osteoclast formation and bone resorption by BMMP or peritoneal exudate cells (PEC). BMMP or PEC were co-cultured with liver or dead bone, i.e., in the presence or absence of liver bone-forming cells. Mineral resorption and osteoclast formation were monitored via 45Ca release from prelabeled live or dead bone followed by histology. Osteoclasts developed from precultured BMMP as indicated by [3H]thymidine labeling, but only in live and not in dead bone. They formed readily from BMMP but only erratically, and after a longer culture period, from PEC. Macrophages from BMMP and PEC invaded live and dead bone rudiments but did not resorb the intact mineralized matrix. In contrast, ground bone powder was resorbed avidly by both cell populations, without formation of osteoclasts. We conclude that live bone-forming cells are required for osteoclast formation from progenitors. Live bone is only resorbed by osteoclasts, and not by macrophages. Osteoclast progenitors are abundant in cultures of BMMP but scarce in PEC, which makes a direct descendance of osteoclasts from mature macrophages unlikely.     

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

The differentiation and activity of osteoclasts are positively and negatively controlled by receptor activator of nuclear factor-kappaB ligand (RANKL), which is expressed on the surface of osteoblasts and stromal cells, and its decoy receptor osteoprotegerin (OPG), which is secreted by osteoblasts and stromal cells, respectively. The expression of the genes for RANKL and OPG is regulated by 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)]. Runt-related gene-2 (Runx-2) is essential for osteoblast differentiation and there are several reports that Runx-2 is involved in osteoclast formation. Therefore, to clarify the role of Runx-2 in osteoclastogenesis, we designed a series of experiments using C2 cells and C6 cells, which are derived from calvariae of runx2-deficient mice. Treatment of C2 cells and C6 cells with 1alpha,25(OH)(2)D(3) for 2-4 days increased and decreased the levels of expression of the mRNAs for RANKL and OPG, respectively, and the effects were dose-dependent. However, by day 8, the level of RANKL mRNA had fallen and that of OPG mRNA had risen. Furthermore, C6 cells induced the differentiation of mouse spleen cells into tartrate-resistant acid phosphatase-positive (TRAP-positive) multinucleated cells (osteoclast-like cells) in the presence of 10(-7)M 1alpha,25(OH)(2)D(3). Such formation of osteoclast-like cells was inhibited by exogenous OPG in a dose-dependent manner. Thus, our findings indicate that Runx-2 is not essential for the expression of RANKL and OPG, and the formation of osteoclast-like cells.     

Human macrophage colony-stimulating factor inhibits bone resorption by osteoclasts disaggregated from rat bone

Colony stimulating factors (CSFs) regulate the survival, proliferation and differentiation of haemopoietic progenitor cells, as well as the functional activity of mature cells. Because the osteoclast is derived from haemopoietic tissue, and because osteoblastic cells produce CSFs, we tested the effects of several CSFs on bone resorption by osteoclasts disaggregated from neonatal rat long bone. We found that recombinant macrophage (M)-CSF was a potent inhibitor of bone resorption, causing significant inhibition at concentrations similar to those required to support the growth of macrophage colonies in agar. Unlike other inhibitors of osteoclastic resorption, M-CSF did not alter cytoplasmic motility in time-lapse recordings, suggesting that M-CSF may inhibit osteoclasts through a different transduction mechanism. None of the remaining cytokines tested (granulocyte-macrophage CSF, interleukin 3, interleukin 6, or interferon γ) influenced bone resorption. M-CSF production may be a mechanism by which osteoblastic cells, which produce M-CSF, may regulate osteoclastic function. Alternatively, inhibition of osteoclastic resorption by a CSF that is responsible for amplification of the macrophage compartment may reflect a close lineage relationship between mononuclear phagocytes, in which M-CSF induces a diversion of lineage resources away from osteoclastic function.     

Mouse strain-dependent osteoclastogenesis in response to lipopolysaccharide

Bacterial lipopolysaccharide (LPS) is a potent stimulator of bone resorption in periodontitis. Co-culture systems of mouse calvaria-derived osteoblasts and bone marrow-derived preosteoclasts were used as an in vitro osteoclast differentiation. This study revealed that co-cultures using ddY or ICR mouse strain responded differently to LPS while responded equally to 1alpha,25(OH)2D3. Thus, the different response to LPS indicates dissimilarity of two mouse stains in their capacity for generating osteoclasts while the two mouse strains share the similarity in response to 1alpha,25(OH)2D3. To identify which cells between osteoblasts and preosteoclasts in the co-culture are responsible for the dissimilarity, the reciprocal co-cultures were performed between ddY and ICR mouse strains. The treatment of 1,25(OH)2D3 to ddY/ICR (osteoblasts from ddY/preosteoclasts from ICR) and ICR/ddY reciprocal co-cultures also showed the similarity. In case of LPS treatment, the results of ddY/ICR were similar to ddY/ddY and the results of the other reciprocal co-culture, ICR/ddY combination, were consistent with those of ICR/ICR. It suggests that the dissimilarity between the two mouse strains may resident in osteoblasts but not in preosteoclasts. Therefore, the osteoblast is responsible for mouse strain-dependent osteoclastogenesis in response to LPS. Although mouse models will continue to provide insights into molecular mechanisms of osteoclastogenesis, caution should be exercised when using different mouse strains, especially ddY and ICR strains as models for osteoclast differentiation.     

1,25-Dihydroxyvitamin D(3) and prostaglandin E(2) act directly on circulating human osteoclast precursors.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and prostaglandin E(2) (PGE(2)) are known to influence osteoclast formation indirectly through their effects on osteoblasts. To determine whether 1, 25(OH)(2)D(3) and PGE(2) also have a direct effect on circulating osteoclast precursors, these factors were added to long-term cultures of human peripheral blood mononuclear cells (PBMCs) in the presence of osteoprotegerin ligand and macrophage colony-stimulating factor (M-CSF) (+/-dexamethasone). The number of TRAP(+) and VNR(+) multinucleated cells and the area of lacunar resorption were decreased when 1,25(OH)(2)D(3) alone was added. A marked increase in resorption pit formation was noted when the combination of 1, 25(OH)(2)D(3) and dexamethasone was added to PBMC cultures. Dose-dependent inhibition of osteoclast formation and lacunar resorption was seen when PGE(2) was added to PBMC cultures in both the presence and the absence of dexamethasone. Thus, 1,25(OH)(2)D(3) and PGE(2) not only influence osteoclast formation in the presence of bone stromal cells but also act directly on circulating osteoclast precursors to influence osteoclast differentiation.     

A novel interaction between thyroid hormones and 1,25(OH)(2)D(3) in osteoclast formation

Thyroid hormones enhance osteoclast formation and their excess is an important cause of secondary osteoporosis. 3,5,3' -Triiodo-L-thyronine (T3) induced the mRNA expression of receptor activator of nuclear factor-kappa B ligand (RANKL), which is a key molecule in osteoclast formation, in primary osteoblastic cells (POB). This effect was amplified in the copresence of 1 alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). Although T3 alone did not induce octeoclasts in coculture of bone marrow cells with POB, T3 enhanced 1,25(OH)(2)D(3)-induced osteoclast formation. Thyroxine (T4) also enhanced 1,25(OH)(2)D(3)-induced osteoclast formation. These data suggested that T4 was locally metabolized to T3 for its action, since T4 is a prohormone with little hormonal activity. The mRNA expression of type-2 iodothyronine deiodinase (D2), which is responsible for maintaining local T3 concentration, was induced by 1,25(OH)(2)D(3) dose- and time-dependently. Our data would facilitate our understanding of the mechanism of osteoclast formation by thyroid hormones and suggest a novel interaction between thyroid hormones and 1,25(OH)(2)D(3).     

IL-4, but not vitamin D(3), induces monoblastic cell line UG3 to differentiate into multinucleated giant cells on osteoclast lineage

The formation of multinucleated giant cells (MGCs) from monocytes/macrophages is controlled by various cytokines, the roles of which are not fully understood. Both interleukin (IL)-4 and 1alpha,25(OH)(2) vitamin D(3) (D(3)) are known to induce MGC formation from monocytes/macrophages. D(3) is also known as a stimulator of osteoclast formation in the presence of stroma cells, and IL-4 as an inhibitor. Previously, we showed that IL-4-induced MGCs from monocytes/macrophages expressed tartrate resistant acid phosphatase (TRAP) activity and hydroxyapatite-resorptive activity in the presence of M-CSF without stroma cells. In this study, we examined the effects of D(3) and/or IL-4 on MGC formation and the characteristics of these MGCs using a monoblastic cell line (UG3), to elucidate the involvement of these factors in osteoclast development without stroma cells. D(3)-induced MGCs showed none of the markers of osteoclasts, such as TRAP activity, calcitonin receptor (cal-R) expression, hydroxyapatite-resorptive activity, and bone-resorptive activity. A low concentration of D(3) synergistically stimulated IL-4-induced TRAP-positive MGC formation, whereas a high concentration of D(3) inhibited it. When IL-4 was added on day 7 of the 2-week culture with D(3), TRAP positivity reached maximum. On the other hand, delayed addition of D(3) on day 7 of culture did not increase the TRAP positivity. Although the fusion rate increased during the first week of the 2-week culture in the presence of D(3), it increased further in the second week following the addition of IL-4 on day 7. Furthermore, IL-4-induced, or IL-4- and D(3)-induced MGCs differentiated into functional osteoclasts with bone-resorptive activity following coculture with osteoblastic cells, whereas D(3)-induced MGCs did not acquire bone-resorptive activity even after coculture with osteoblastic cells in the presence of D(3). These findings suggest that IL-4 initiates osteoclast development of UG3 cells, although stroma cells were necessary for development of functional osteoclasts. On the other hand, D(3) had only a "supportive" effect on this differentiation. IL-4 and direct contact with stroma cells may regulate different stages in the multistep process of osteoclastogenesis of UG3 cells.