The synthetic triterpenoid TP-222 inhibits RANKL stimulation of osteoclastogenesis and matrix metalloproteinase-9 expression

OBJECTIVE: Receptor activator of nuclear factor-kappaB ligand (RANKL) promotes osteoclast differentiation from monocyte precursors by inducing a cohort of genes, including tartrate-resistant acid phosphatase (TRAP) and matrix metalloproteinase-9 (MMP-9). A family of synthetic triterpenoids with antiinflammatory and pro-apoptotic properties was described to modulate differentiation in monocytic cell lineages. We therefore investigated the ability of the potent and bioavailable synthetic triterpenoid TP-222 to inhibit RANKL-induced osteoclast formation and MMP-9 expression from monocytic precursor cells. METHODS: Osteoclast formation was assayed by staining for TRAP-positive multinucleated cells. MMP-9 expression was measured by quantitative RT-PCR, Western blot, immunohistochemistry, and gel zymography. In vivo effects of TP-222 were assessed by daily intraperitoneal injection of 4-week-old mice for 7 days followed by measurement of osteoclast number and MMP-9 expression at the cartilage/bone junction of the epiphyseal growth plate. RESULTS: RANKL promoted and TP-222 (300 nM) inhibited osteoclast formation in cultures of RAW264.7 cells or bone marrow-derived monocytes. RANKL also induced MMP-9 expression in RAW264.7 cells and this was reduced by concurrent or subsequent addition of TP-222. TP-222 treatment significantly reduced the mean number of osteoclasts present at the cartilage/bone interface compared to vehicle-injected control mice. Morphometric analyses of tissue sections showed that TP-222 treatment reduced the amount of immunoreactive MMP-9 present in both mononucleated pre-osteoclasts and osteoclasts. CONCLUSION: Our data demonstrate that TP-222 inhibits osteoclast formation and MMP-9 expression in vitro and in vivo, and suggest that triterpenoids may be useful compounds for modulating bone resorption diseases.     

RANKL诱导破骨细胞前体细胞分化成熟

目的 用核因子-κB受体活化因子配体(RANKL)诱导破骨细胞前体细胞分化成熟,建立获取成熟破骨细胞的方法.方法 用破骨细胞前体细胞RAW264.7细胞为模型,RANKL诱导培养4～9 d,抗酒石酸酸性磷酸酶(TRAP)染色观察TRAP阳性多核细胞形成,罗丹明-鬼笔环肽荧光染色观察纤维性肌动蛋白(F-actin)环,DAPI染色观察细胞核,甲苯胺蓝染色观察牛骨片表面的吸收陷窝情况.结果 RANKL可诱导RAW264.7细胞形成TRAP染色阳性的多核细胞,形成F-actin环,骨片吸收陷窝明显.结论 RANKL可诱导RAW264.7细胞向成熟破骨细胞分化,该诱导模型可用于破骨细胞分化研究.     

Enhanced osteoclastogenesis in patients with tophaceous gout: urate crystals promote osteoclast development through interactions with stromal cells

OBJECTIVE: To analyze cellular mechanisms of bone erosion in gout. METHODS: Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) from patients with gout were analyzed for the presence of osteoclast precursors. Fixed tophus and bone samples were analyzed by immunohistochemistry. Mechanisms of osteoclastogenesis were studied by culturing murine preosteoclast RAW 264.7 cells, bone marrow stromal ST2 cells, and human synovial fibroblasts with monosodium urate monohydrate (MSU) crystals. RESULTS: PBMCs from patients with severe erosive gout had the preferential ability to form osteoclast-like cells in culture with RANKL and monocyte colony-stimulating factor (M-CSF). The number of PBMC-derived tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells strongly correlated with the number of tophi (r = 0.6296, P = 0.630). Patients with severe erosive and tophaceous gout also had higher circulating concentrations of RANKL and M-CSF. Furthermore, greater numbers of TRAP-positive multinucleated cells were cultured from SFMCs derived from gouty knee effusions than from paired PBMCs (P = 0.004). Immunohistochemical analysis demonstrated numerous multinucleated cells expressing osteoclast markers within tophi and at the interface between soft tissue and bone. MSU crystals did not directly promote osteoclast formation from RAW 264.7 cells in vitro. However, MSU crystals inhibited osteoprotegerin gene and protein expression in ST2 cells and human synovial fibroblasts, without significantly altering RANKL gene expression. Conditioned medium from ST2 cells cultured with MSU crystals promoted osteoclast formation from RAW 264.7 cells in the presence of RANKL. CONCLUSION: Chronic tophaceous and erosive gout is characterized by enhanced osteoclast development. These data provide a rationale for the study of osteoclast-targeted therapies for the prevention of bone damage in chronic gout.     

Direct stimulation of osteoclastogenesis by MIP-1alpha: evidence obtained from studies using RAW264 cell clone highly responsive to RANKL

Macrophage inflammatory protein-1alpha (MIP-1alpha) is a member of the CC chemokines. We have previously reported the use of a whole bone marrow culture system to show that MIP-1alpha stimulates the formation of osteoclast-like multinucleated cells. Here we use rat bone marrow cells deprived of stromal cells, and clones obtained from murine macrophage-like cell line RAW264 to show that MIP-1alpha acts directly on cells in osteoclast lineage. We obtained several types of RAW264 cell clones, one of these clones, designated as RAW264 cell D clone (D clone), showed an extremely high response to receptor activator of NFkappaB ligand (RANKL) and tumor necrosis factor-alpha (TNF-alpha), while the other clone, RAW264 cell N clone (N clone), demonstrated no response to RANKL or TNF-alpha. Although both clones expressed receptor activator NFkappaB (RANK) before being stimulated for differentiation, only the D clone expressed cathepsin K when cells were stimulated to differentiate to osteoclasts. MIP-1alpha stimulated the formation of mononuclear preosteoclast-like cells from rat bone marrow cells deprived of stromal cells. MIP-1alpha also stimulated formation of osteoclast-like multinucleated cells from the D clone, when these cells were stimulated with RANKL and TNF-alpha. These findings provide strong evidence to show that MIP-1alpha acts directly on cells in the osteoclast lineage to stimulate osteoclastogenesis. Furthermore, pretreatment of RAW264 cell D clone with MIP-1alpha significantly induced adhesion properties of these cells to primary osteoblasts, suggesting a crucial role for MIP-1alpha in the regulation of the interaction between osteoclast precursors and osteoblasts in osteoclastogenesis.     

Generation of human osteoclasts in stromal cell-free and stromal cell-rich cultures: differences in osteoclast CD11c/CD18 integrin expression

Osteoclasts form in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of Nfkappab ligand (RANKL), a membrane-bound differentiation factor that is now available as a soluble recombinant molecule. Acquisition of the osteoclast phenotype [the alphavbeta3 subunit of the vitronectin receptor (VNR)-, calcitonin receptor (CTR)- and F-actin ring-positive cells] is associated with loss of monocyte/macrophage-associated integrins, specifically CD11b, CD11c and CD18. We hypothesized that differences in the osteoclast integrin adhesion molecule profile may exist in osteoclasts generated in stromal cell-rich and in stromal-free conditions. Unlike osteoclasts generated in vivo, F-actin ring-positive (resorbing) osteoclasts formed in soluble RANKL in vitro, in the absence of stromal cells, and co-expressed CD11c and CD18. However, when osteoclasts were generated from peripheral blood mononuclear cells (PBMNCs) in co-cultures with the murine bone marrow stromal cell line 218 (which does not produce membrane-bound RANKL) in the presence of soluble RANKL, CD11c and CD18 were not expressed by osteoclasts. These findings indicate that the persistent expression of CD11c and CD18 is not accounted for by RANKL being presented in a soluble form and that membrane-bound RANKL is not required for the normal integrin expression in resorbing osteoclasts. This study demonstrates that potentially misleading information may arise by using data obtained from osteoclasts generated in the absence of stromal cells as they do not completely reflect the situation in vivo.     

Joint-protective effects of compound K, a major ginsenoside metabolite, in rheumatoid arthritis: in vitro evidence

Regulatory expression of matrix metalloproteinases (MMPs) and osteoclastogenesis is implicated in the process of joint destruction in rheumatoid arthritis (RA). Although several reports suggested the anti-arthritic effects of ginseng saponins, it has not been investigated whether the most absorbable ginsenoside, compound K (CK), has a joint-protective action. We here investigated the effect of CK (0–5 μM) on TNF-α-induced MMP-1, MMP-3, and MMP-13 and TIMP-1 production from RA fibroblast-like synoviocytes (FLS) and determined the inhibitory effect of CK on osteoclastogenesis from RAW264.7 cells co-cultured with RA-FLS and from human CD14+ monocytes. The effect of CK on NF-κB, nuclear factor of activated T cells c1 (NFATc1), and mitogen-activated protein kinases pathways were evaluated using immunoblotting or specific inhibitors. CK significantly inhibited MMP-1 and MMP-3 productions from RA-FLS in a concentration-dependent manner through suppressing the JNK and ERK pathways. In the co-culture system of TNF-α-stimulated RA-FLS and RAW264.7 cells, CK dose-dependently reduced receptor activator of NF-κB ligand (RANKL) expression in the RA-FLS and inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells. Furthermore, CK significantly inhibited soluble RANKL-induced osteoclastogenesis or osteoclast activity in RAW264.7 cells and human CD14+ monocytes through inhibition of RANKL-induced IκBα degradation and NFATc1 expression. In conclusion, our results increase the understanding of the molecular mechanisms of the joint-protective effects of CK in RA. The characteristic actions of CK provide in vitro evidence for its potential utility in RA therapy.     

Mechanisms involved in bone resorption

Osteoclasts, which are present only in bone, are multinucleated giant cells with the capacity to resorb mineralized tissues. These osteoclasts are derived from hemopoietic progenitors of the monocyte-macrophage lineage. Osteoblasts or bone marrow-derived stromal cells are involved in osteoclastogenesis through a mechanism involving cell-to-cell contact with osteoclast progenitors. Experiments on the osteopetrotic op/op mouse model have established that a product ofosteo blasts, macrophage colony-stimulating factor (M-CSF), regulates differentiation of osteoclast progenitors into osteoclasts. Recent discovery of osteoclast differentiation factor (ODF)/receptor activator of NF-κ Bligand (RANKL) allowed elucidation of the precise mechanism by which osteoblasts regulate osteoclastic bone resorption. Treatment of osteoblasts with bone-resorbing factors up-regulated expression of RANKL mRNA. In contrast, TNF α stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the RANKL system. IL-1 also directly acts on mature osteoclasts as a potentiator of osteoclast activation. In addition, TGF-β super family members, such as bone morphogenetic proteins(BMPs) strikingly enhanced osteoclast differentiation from their progenitors and survival of mature osteoclasts induced by RANKL. These results suggest that BMP-mediated signals cross-communicate with RANKL-mediated ones in inducing osteoclast differentiation and function. This revised version was published online in July 2006 with corrections to the Cover Date.     

Role of FK506 binding protein 5 (FKBP5) in osteoclast differentiation

Objectives

We previously disclosed the enhanced expression of FK506 binding protein 5 (FKBP5) messenger RNA (mRNA) in bone marrow (BM) CD34⁺ cells in rheumatoid arthritis (RA), in which systemic osteoporosis takes place. Since BM CD34⁺ cells are precursors of osteoclasts, it is possible that FKBP5 overexpression might lead to osteoporosis by affecting osteoclastogenesis. We therefore explore the influences of FKBP5 in osteoclast differentiation.

Methods

Stable transfectants of RAW264.7 overexpressing murine FKBP5 gene were established. Osteoclast differentiation was induced by receptor activator of nuclear factor kappa B (NF-κB) ligand and was evaluated by tartrate-resistant acid phosphatase (TRAP) staining and pit formation assay.

Results

FKBP5 transfectants of RAW264.7 generated higher numbers of TRAP-positive multinucleated cells with increased activity of pit formation on calcium phosphate-coated culture slides than mock transfectants. The enhancement of osteoclast differentiation of FKBP5 transfectants was only partially inhibited by N-acetyl l-cysteine. Finally, glucocorticoid enhanced FKBP5 mRNA expression as well as osteoclast differentiation of RAW264.7 cells in a dose-dependent manner.

Conclusions

These results indicate that FKBP5 promotes osteoclast differentiation by a mechanism distinct from NF-κB activation. Moreover, the data suggest that FKBP5 might play a role in bone destruction and development of osteoporosis in RA as well as in glucocorticoid-induced osteoporosis.     

Abundant expression of tetraspanin CD9 in activated osteoclasts in ovariectomy-induced osteoporosis and in bone erosions of collagen-induced arthritis

Tetraspanin CD9 has been shown to be critically involved in multinucleation and cell fusion during osteoclastogenesis, however, its in vivo pathophysiological role in bone-resorbing disorders such as osteoporosis and rheumatoid arthritis, has not been elucidated. To investigate the involvement of tetraspanin CD9 in bone destruction in such diseases, we examined the expression and distribution of tetraspanin CD9 using murine experimental models of osteoporosis and arthritis. In results, CD9 protein is abundantly expressed on the activated osteoclasts in the bone tissues whose trabeculae are severely reduced in ovariectomy-induced osteoporosis. The expression of CD9 is also detected at the sites of bone erosion in arthritic lesions of collagen-induced arthritis (CIA), where tartate-resistant acid phosphatase (TRAP) staining-positive activated osteoclasts are present. These data suggest that tetraspanin CD9 play important roles in bone destructions in osteoporosis and arthritis, and therefore, functional alterations of tetraspanin CD9 may have therapeutic potential in such bone-resorptive disorders.     

Lipopolysaccharide-induced osteoclastogenesis in Src homology 2-domain phosphatase-1-deficient viable motheaten mice

Osteoclasts are hemopoietic cells that participate in bone resorption and remodeling. Receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) are critical for development of osteoclasts. The Toll-like receptor (TLR) family shares some of the downstream signaling with RANK. The TLR4 ligand, lipopolysaccharide (LPS), is reported to accelerate bone lysis; however, signaling via TLRs has never been reported to induce osteoclastogenesis without RANKL. In this study we showed that significant numbers of mature osteoclasts were generated from protein tyrosine phosphatase Src homology 2-domain phosphatase-1-defective Hcph(me-v)/Hcph(me-v) (me(v)/me(v)) bone marrow cells in the presence of M-CSF and LPS without addition of RANKL in culture. This M-CSF plus LPS-induced osteoclastogenesis was not inhibited by an anti-TNFalpha antagonistic antibody or by osteoprotegerin, a decoy receptor for RANKL. The replacement of RANKL by TLR ligands only occurred with LPS. Other ligands, a peptidoglycan for TLR2 or an unmethylated CpG oligonucleotide for TLR9, did not support osteoclast generation. The osteoclast precursors as well as RANKL-responsive osteoclast precursors were present in the Kit-positive cell-enriched fraction of bone marrow cells. Although me(v)/me(v) bone marrow cells required a comparable concentration of RANKL or TNFalpha as wild-type cells for the initiation of osteoclastogenesis, the numbers of multinucleated osteoclasts in me(v)/me(v) bone marrow cultures were significantly increased by the equivalent dose of RANKL or TNFalpha in the presence of M-CSF. These results indicate that a defect of Src homology 2-domain phosphatase-1 function not only accelerates physiological osteoclast development by RANKL/RANK, but also acquires a novel pathway for osteoclastogenesis by LPS.     

The role of calmodulin in the regulation of osteoclastogenesis

Calmodulin plays an important role in regulating the function of mature osteoclasts. However, its role in osteoclastogenesis has not been investigated. In the present study, we examined the role of calmodulin in osteoclastogenesis using in vivo and in vitro systems. Calmodulin antagonists, trifluoperazine (TFP), W7, and tamoxifen, dose-dependently inhibited osteoclast formation, which occurred only in the last 24 h of a 4-d osteoclastogenesis culture using mouse bone marrow macrophages. Inhibitory effects were quantitated by measuring tartrate-resistant acid phosphatase activity and counting osteoclast numbers. In contrast, bis indolylmaleimide, a protein kinase C inhibitor, showed no such inhibitory effect even when applied at a concentration that was 10-fold greater than its IC50. Overexpressing calmodulin by recombinant retrovirus reversed the inhibitory effect of TFP on osteoclast-like differentiation in RAW264.7 cells. Furthermore, administration of TFP to mice was as effective as estrogen in abolishing the ovariectomy-induced increment of osteoclastogenesis as determined by quantitative assessment of tartrate-resistant acid phosphatase activity in tibias, which led to the recovery of the ovariectomy-induced decrement in trabecular bone volume. To investigate potential cellular and molecular mechanisms by which calmodulin antagonists inhibit osteoclastogenesis, Z-VAD-FMK, a broad caspase inhibitor, failed to block the inhibitory effect of TFP on mouse osteoclast formation, indicating that apoptosis is not the underlying mechanism. Pretreatment of RAW264.7 cells with different concentrations of TFP dose-dependently inhibited receptor activator of nuclear factor kappaB ligand-stimulated phosphorylation of c-Jun N-terminal kinase and inhibitory kappaBalpha but not that of p38. Taken together, our data indicate that calmodulin mediates osteoclast differentiation, possibly via modulating specific receptor activator of NF-kappaB-signaling pathways.