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.     

Bone morphogenetic protein 2 stimulates osteoclast differentiation and survival supported by receptor activator of nuclear factor-kappaB ligand.

Bone is a major storage site for TGFbeta superfamily members, including TGFbeta and bone morphogenetic proteins. It is believed that these cytokines are released from bone during bone resorption. Recent studies have shown that both RANKL and macrophage colony-stimulating factor are two essential factors produced by osteoblasts for inducing osteoclast differentiation. In the present study we examined the effects of bone morphogenetic protein-2 on osteoclast differentiation and survival supported by RANKL and/or macrophage colony-stimulating factor. Mouse bone marrow-derived macrophages differentiated into osteoclasts in the presence of RANKL and macrophage colony-stimulating factor. TGFbeta superfamily members such as bone morphogenetic protein-2, TGFbeta, and activin A markedly enhanced osteoclast differentiation induced by RANKL and macrophage colony-stimulating factor, although each cytokine alone failed to induce osteoclast differentiation in the absence of RANKL. Addition of a soluble form of bone morphogenetic protein receptor type IA to the culture markedly inhibited not only osteoclast formation induced by RANKL and bone morphogenetic protein-2, but also the basal osteoclast formation supported by RANKL alone. Either RANKL or macrophage colony-stimulating factor stimulated the survival of purified osteoclasts. Bone morphogenetic protein-2 enhanced the survival of purified osteoclasts supported by RANKL, but not by macrophage colony-stimulating factor. Both bone marrow macrophages and mature osteoclasts expressed bone morphogenetic protein-2 and bone morphogenetic protein receptor type IA mRNAs. An EMSA revealed that RANKL activated nuclear factor-kappaB in purified osteoclasts. Bone morphogenetic protein-2 alone did not activate nuclear factor-kappaB, but rather inhibited the activation of nuclear factor-kappaB induced by RANKL in purified osteoclasts. These findings suggest that bone morphogenetic protein-mediated signals cross-communicate with RANKL-mediated ones in inducing osteoclast differentiation and survival. The enhancement of RANKL-induced survival of osteoclasts by bone morphogenetic protein-2 appears unrelated to nuclear factor-kappaB activation.     

Expression and role of mannose receptor/terminal high-mannose type oligosaccharide on osteoclast precursors during osteoclast formation

Osteoclasts are formed from hematopoietic precursors via cell-cell fusion. We have previously reported that mannose residues are expressed on the outer membranes of monocytes during osteoclast differentiation. In the present study, we have attempted to demonstrate the pattern of expression levels of terminal high-mannose type oligosaccharide and to show that the mannose receptor is expressed on osteoclast precursor cells. Osteoclasts were formed using three different systems, namely mouse bone marrow cell culture, co-culture of mouse spleen cells with stromal cells, and RAW264.7 cell cultures. During osteoclast differentiation, the expression of terminal high-mannose type oligosaccharide gradually increased and then peaked at the stage of fusion in all three systems. Expression of the mannose receptor gradually increased during osteoclast differentiation in bone marrow cells and the co-culture system. In contrast, that in RAW264.7 cells had already been detected in the absence of the soluble receptor activator of NF-kappaB ligand and did not change during osteoclast differentiation. To ascertain whether expression of high-mannose type oligosaccharide is involved in tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cell (MNC) formation, glycosidase inhibitors were used on RAW264.7 cell culture. Castanospermine, an inhibitor of glucosidase I, inhibited the TRAP-positive MNCs, and deoxymannojirimycin, an inhibitor of alpha-mannosidase I, increased the TRAP-positive MNC formation. These results indicate that the binding of terminal high-mannose and mannose receptor is important for the process of cellular fusion in osteoclast formation.     

Ascorbic acid inhibits osteoclastogenesis of RAW264.7 cells induced by receptor activated nuclear factor kappaB ligand (RANKL) in vitro

Ascorbic acid (AA) plays a key role in the regulation of differentiation and activation of osteoclast (OCL). It was reported that AA might induce the formation of OCL in cocultures of mouse bone marrow cells and ST2 cells, but it is not clear whether AA has a direct impact on the OCL precursors. The purpose of this study was to examine the effect of AA on the differentiation of OCL precursor RAW264.7 cells, cultured with receptor-activated nuclear factor kappaB ligand (RANKL). The results showed that AA remarkably inhibited the cell proliferation at a higher concentration and RANKL alone is sufficient for osteoclastogenesis. The expression of carbonic anhydrase (CAII) mRNA and protein, the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), and the percentage area of resorption lacunae induced by RANKL were decreased when AA was added to the cultures. The results demonstrate that AA inhibits RANKL-induced differentiation of OCL precursor cells into mature OCL and reduces the formation of bone resorption pits in vitro.     

Hydrogen peroxide is essential for estrogen-deficiency bone loss and osteoclast formation

We recently found that estrogen deficiency leads to a lowering of thiol antioxidant defenses in rodent bone. Moreover, administration of agents that increase the concentration in bone of glutathione, the main intracellular antioxidant, prevented estrogen-deficiency bone loss, whereas depletion of glutathione by buthionine sulfoximine administration provoked substantial bone loss. To analyze further the mechanism by which antioxidant defenses modulate bone loss, we have now compared expression of the known antioxidant enzymes in osteoclasts. We found that glutathione peroxidase 1 (Gpx), the enzyme primarily responsible for the intracellular degradation of hydrogen peroxide, is overwhelmingly the predominant antioxidant enzyme expressed by osteoclasts and that its expression was increased in bone marrow macrophages by receptor activator of nuclear factor-kappaB ligand (RANKL) and in osteoclasts by 17beta-estradiol. We therefore tested the effect of overexpression of Gpx in osteoclasts by stable transfection of RAW 264.7 (RAW) cells, which are capable of osteoclastic differentiation in response to RANKL, with a Gpx-expression construct. Osteoclast formation was abolished. The Gpx expression construct also suppressed RANKL-induced nuclear factor-kappaB activation and increased resistance to oxidation of dihydrodichlorofluorescein by exogenous hydrogen peroxide. We therefore tested the role of hydrogen peroxide in the loss of bone caused by estrogen deficiency by administering pegylated catalase to mice. We found that catalase prevented ovariectomy-induced bone loss. These results suggest that hydrogen peroxide is the reactive oxygen species responsible for signaling the bone loss of estrogen deficiency.     

Transcriptional induction of cyclooxygenase-2 in osteoclast precursors is involved in RANKL-induced osteoclastogenesis

Regulation of osteoclast differentiation is key to understanding the pathogenesis and to developing treatments for bone diseases such as osteoporosis. To gain insight into the mechanism of the receptor activator of nuclear factor (NF)-kappaB ligand (RANKL)-specific induction of the osteoclast differentiation program, we took a suppression-subtractive hybridization screening approach to identify genes specifically induced via the RANKL-Rac1 signaling pathway. Among identified targets, we show that RANKL selectively induces cyclooxygenase (COX) 2 expression via Rac1 that results in turn in production of prostaglandin E2 (PGE2) in RAW 264.7 cells. By using transient transfection assays, we found that the -233/-206 region of the COX-2 promoter gene was critical for RANKL-induced promoter activity. This RANKL-responsive region contained an NF-kappaB site that, when mutated, completely abolished the induction of NF-kappaB DNA-binding activity by RANKL. Blockade of COX-2 by celecoxib inhibits differentiation of bone marrow-derived monocyte/macrophage precursor cells (BMMs) into tartrate-resistant acid phosphatase-positive (TRAP+) osteoclastic cells. This inhibition can be rescued by the addition of exogenous PGE2, suggesting that COX-2-dependent PGE2 induction by RANKL in osteoclast precursors is required for osteoclast differentiation.     

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.     

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.     

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.     

p38 MAPK-mediated signals are required for inducing osteoclast differentiation but not for osteoclast function

Receptor activator of nuclear factor-kappaB ligand (RANKL)-induced signals play critical roles in osteoclast differentiation and function. SB203580, an inhibitor of p38 MAPK, blocked osteoclast formation induced by 1alpha,25-dihydroxyvitamin D(3) and prostaglandin E(2) in cocultures of mouse osteoblasts and bone marrow cells. Nevertheless, SB203580 showed no inhibitory effect on RANKL expression in osteoblasts treated with 1alpha,25-dihydroxyvitamin D(3) and prostaglandin E(2). RANKL-induced osteoclastogenesis in bone marrow cultures was inhibited by SB203580, suggesting a direct effect of SB203580 on osteoclast precursors, but not on osteoblasts, in osteoclast differentiation. However, SB203580 inhibited neither the survival nor dentine-resorption activity of osteoclasts induced by RANKL. Lipopolysaccharide (LPS), IL-1, and TNFalpha all stimulated the survival of osteoclasts, which was not inhibited by SB203580. Phosphorylation of p38 MAPK was induced by RANKL, IL-1, TNFalpha, and LPS in osteoclast precursors but not in osteoclasts. LPS stimulated phosphorylation of MAPK kinase 3/6 and ATF2, upstream and downstream signals of p38 MAPK, respectively, in osteoclast precursors but not in osteoclasts. Nevertheless, LPS induced degradation of IkappaB and phosphorylation of ERK in osteoclasts as well as in osteoclast precursors. These results suggest that osteoclast function is induced through a mechanism independent of p38 MAPK-mediated signaling.     

铁超载对小鼠单核细胞RAW264．7向破骨细胞分化的影响

]目的探讨铁超载对小鼠单核细胞RAW264．7向成熟破骨细胞分化的影响。方法实验分为正常对照组、低铁对照组、高铁干预组3组,用含有核因子KB受体活化因子配体（RANKL）和巨噬细胞集落刺激因子（M—CSF）的培养基诱导RAW264．7向破骨细胞分化,在此过程中加入去铁胺（DFO）和不同浓度的枸橼酸铁铵（FAC）。对培养第4天和第7天的细胞进行抗酒石酸酸性磷酸酶（TRAP）染色,对TRAP阳性细胞进行计数,用酶联免疫吸附法（ELISA）检测培养基中TRAP-Sb的含量。结果（1）高铁干预组的TRAP阳性细胞数高于正常对照组和低铁对照组（P〈0．05）,具有时间和浓度依赖性;（2）高铁干预组的TRAP-Sb的含量高于正常对照组（P〈0．05）,具有浓度依赖性。结论铁超载能促进RAW264．7向破骨细胞分化。     

The differential expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL) in human osteoarthritic subchondral bone osteoblasts is an indicator of the metabolic state of these disease cells

OBJECTIVE: We previously reported that human OA subchondral bone osteoblasts could be discriminated into two subpopulations identified by their levels of endogenous production (low [L] or high [H]) of PGE(2). Here, we investigated the OPG and RANKL expression levels, the histologic analysis of the subchondral bone as well as the osteoclast differentiation effect of osteoblasts on normal and both OA subpopulations (L and H), and further examined on the L OA osteoblasts the modulation of bone remodelling factors on the OPG and RANKL levels, as well as on the resorption activity. METHODS: Gene expression was determined using real-time PCR, PGE2 and OPG levels by specific ELISA, and membranous RANKL by flow cytometry. Histological observation of the subchondral bone was performed on human knee specimens. Osteoclast differentiation and formation was assayed by using the pre-osteoclastic cell line RAW 264.7. OPG and RANKL modulation on L OA osteoblasts was monitored following treatment with osteotropic factors, and the resorption activity was studied by the co-culture of differentiated PBMC/osteoblasts. RESULTS: Human OA subchondral bone osteoblasts expressed less OPG than normal. Compared to normal, RANKL gene expression levels were increased in L OA and decreased in H OA cells. The OPG/RANKL mRNA ratio was significantly diminished in L OA compared to normal or H OA (p<0.02, p<0.03), and markedly increased in H OA compared to normal. Inhibition of endogenous PGE(2) levels by indomethacin markedly decreased the ratio of OPG/RANKL on the H OA. In contrast to H OA osteoblasts, L OA cells induced a significantly higher level of osteoclast differentiation and formation (p<0.05).Histological analysis showed a reduced subchondral bone on the L OA and an increased bone mass on the H OA compared to normal. Treatment of L OA osteoblasts with osteotropic factors revealed that the OPG/RANKL mRNA expression ratio was significantly reduced by vitamin D(3) and significantly increased by TNF-alpha, PTH and PGE(2), while IL-1Beta demonstrated no effect. OPG protein levels showed similar profiles. No true effect was noted on membranous RANKL upon treatment with IL-1Beta, PGE(2) and PTH, but a significant increase was observed with vitamin D3 and TNF-alpha. The resorption activity of the L OA cells was significantly inhibited by all treatments except IL-1Beta, with maximum effect observed with vitamin D(3) and PGE(2). CONCLUSION: OPG and RANKL levels, and consequently the OPG/RANKL ratio, differed according to human OA subchondral bone osteoblast classification; it is decreased in L and increased in H OA. These findings, in addition to those showing that L OA osteoblasts have a reduced subchondral bone mass and induce a higher level of osteoclast differentiation, strongly suggest that the metabolic state of the L OA osteoblasts favours bone resorption.