骨巨细胞瘤中骨保护因子及破骨细胞分化因子的表达

目的 检测骨巨细胞瘤中骨保护因子（OPG）及破骨细胞分化因子（ODF）mRNA的表达,并探讨多核巨细胞的来源及造成骨吸收的分子机制。方法 采用半定量逆转录－聚合酶链反应（RT－PCR）方法检测骨巨细胞瘤和正常骨组织中OPG、ODF及其辅助因子巨噬细胞克隆刺激因子、信号受体RANK mRNA的表达,并将两种组织中各因子的表达情况进行半定量比较。结果 正常骨组织中OPG、巨噬细胞克隆刺激因子、RANK mRNA有表达,ODF呈微弱表达;骨巨细胞瘤中OPG、ODF、巨噬细胞克隆刺激因子、RANK mRAN均有表达,ODF的表达非常丰富。骨巨细胞瘤中ODF与OPG的比值远远高于正常骨组织。结论 骨巨细胞瘤中的微环境具备破骨细胞形成及其促进骨吸收的必要条件,多核巨细胞的来源及其骨吸收功能与上述几种因子的表达有关。     

Hepatocellular carcinoma with osteoclast-like giant cells: possibility of osteoclastogenesis by hepatocyte-derived cells

Giant cell tumor (GCT) of bone is a primary osteolytic tumor that is characterized by the formation of osteoclast-like giant cells. In addition to GCT of bone, extraskeletal GCT are known to be formed in several soft tissues. Giant cells in GCT of bone were suggested to be identical to osteoclasts, but the characterization of giant cells in extraskeletal GCT remains incomplete. In this study, a case of sarcomatoid hepatocellular carcinoma with osteoclast-like giant cells was analyzed. Immunohistochemistry revealed the expression of almost all markers of osteoclasts: tartrate-resistant acid phosphatase, CD68, CD51, CD54 and matrix metalloprotease-9, in osteoclast-like giant cells in the tumor. In situ hybridization revealed the expression of receptor activator of nuclear factor-kappa B (RANK) in the giant cells and receptor activator of nuclear factor-kappa B ligand (RANKL) in the tumor cells. The hepatic origin of the sarcomatoid hepatocellular carcinoma cells was confirmed by the expression of albumin. This is the first report suggesting that hepatocyte-derived cells possess the potential for osteoclastogenesis. In addition, these findings suggest that osteoclast-like cells in the hepatocellular carcinoma were formed by the same mechanism as osteoclastogenesis in bone.     

Rheumatoid arthritis synovial macrophage-osteoclast differentiation is osteoprotegerin ligand-dependent

Osteoprotegerin ligand (OPGL) is a newly discovered molecule which is essential for osteoclast differentiation. Both OPGL and its soluble decoy receptor, osteoprotegerin (OPG), which inhibits osteoclast formation, are known to be produced by osteoblasts and inflammatory cells found in the rheumatoid arthritis (RA) synovium. In this study, RA synovial macrophages were incubated in the presence or absence of OPGL, macrophage-colony stimulating factor (M-CSF), and dexamethasone for various time points. The results indicated that osteoclast formation from RA synovial macrophages is OPGL-dependent and that OPGL and M-CSF are the only humoral factors required for RA synovial macrophage-osteoclast differentiation. OPG was found to inhibit osteoclast formation by RA synovial macrophages in a dose-dependent manner. This study has shown that macrophages isolated from the synovium of RA patients are capable of differentiating into osteoclastic bone-resorbing cells; this process is OPGL- and M-CSF-dependent and is modulated by corticosteroids. Cellular (T and B cells, dendritic cells) and humoral factors in RA synovium and bone may influence osteoclast formation and bone resorption by controlling OPGL/OPG production.     

Detection of mRNAs for urokinase-type plasminogen activator, its receptor, and type 1 inhibitor in giant cell tumors of bone with in situ hybridization.

Although giant cell tumor of bone (GCT) is generally considered to be an uncommon benign neoplasm, it can pursue an aggressive course with local recurrence and metastasis. Attempts to predict the biological behavior of GCT with histopathological parameters, however, have not been successful. The urokinase-type plasminogen activation system has been implicated in tumor invasion and metastasis and abnormalities of the components of this system have been found in several malignancies. In this study we postulated that the urokinase-type plasminogen activation system associated with bone destruction and local invasion is present in GCT. We therefore evaluated the mRNA levels for urokinase-type plasminogen activator (u-PA), urokinase-type plasminogen activator receptor (u-PAR), and plasminogen activator inhibitor type 1 (PAI-1) by using Northern blot analysis and in situ hybridization in four cases of GCT and spindle-shaped mononuclear cells at the 35th passage from a GCT. Our results showed that giant cell tumors of bone contained variable levels of u-PA, u-PAR, and PAI-1 mRNA, respectively, 2.3, 1.4, and 3.2 kb in size. In situ hybridization showed that u-PA, u-PAR, and PAI-1 mRNA were expressed in both the mononuclear cells and the osteoclast-like giant cells; the signal for u-PA mRNA in the spindle-shaped mononuclear cells was more intense than that in the osteoclast-like multinuclear giant cells. Some spherical mononuclear cells (macrophage-like cells) expressed high levels of PAI-1 mRNA in comparison with the spindle-shaped mononuclear cells. In addition, the 35th passaged spindle-shaped mononuclear cells were used to study the gene expression of u-PA during cell proliferation. The results showed that the level of u-PA mRNA increases after adding 10% fetal calf serum to quiescent cells. The induction was maximal at 16 hours and remained high during 48 hours of treatment. In conclusion, even though osteoclast-like cells are ultimately responsible for the bone resorption of GCT, the mononuclear neoplastic cells of GCT may also be involved in degradation of the extracellular matrix during invasive growth by facilitating the urokinase plasminogen activation system. In addition, our observation of upregulation of u-PA mRNA in spindle-shaped mononuclear cells after serum stimulation indicated that u-PA production may be linked to tumor growth.     

Suppression of polyethylene particle-induced osteolysis by exogenous osteoprotegerin

Alterations of the key regulators of osteoclastogenesis, receptor activator of NF-kappaB (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG) have been implicated in wear particle-induced osteolysis, the most common cause for implant failure in total joint replacements. This study investigated the effect of exogenous OPG on ultra-high-molecular-weight polyethylene (UHMWPE) particle-induced osteolysis. The murine calvarial osteolysis model was utilized in 28 C57BL/6J mice randomized to four groups. Group I underwent sham surgery only, group II received UHMWPE particles, and group III and IV particles and subcutaneous OPG starting from day 0 (group III) or day 5 (group IV) until sacrifice. After 2 weeks, calvaria were prepared for histology and histomorphometry. Bone resorption was measured within the midline suture using Giemsa staining and osteoclast numbers were determined using TRAP staining. UHMWPE particle implantation resulted in grossly pronounced osteoclastogenesis and bone resorption. Both immediate and delayed treatment with OPG counteracted these particle-induced effects significantly, suppressing osteoclast formation and bone resorption (p < 0.001 and p < 0.001, respectively). In conclusion, exogenous OPG markedly suppressed UHMWPE particle-induced osteolysis in a murine calvarial model. This important finding underscores the crucial significance of the OPG-RANKL-RANK signaling in wear particle-induced osteolysis. Exogenous OPG may prove an effective treatment modality for wear debris-mediated periprosthetic osteolysis after total joint arthroplasty.     

Expression of osteoprotegerin and RANK ligand in breast cancer bone metastasis

Bone destruction is primarily mediated by osteoclastic bone resorption, and cancer cells stimulate the formation and activation of osteoclasts next to metastatic foci. Accumulating evidences indicate that receptor activator of NF-kB ligand (RANKL) is the ultimate extracellular mediator that stimulates osteoclast differentiation into mature osteoclasts. In contrast, osteoprotegerin (OPG) inhibits osteoclast development. In order to elucidate a mechanism for cancer-induced osteoclastogenesis, cells from a human breast cancer line, MDA-MB-231, were directly co-cultured with ST2, MC3T3-E1, or with primary mouse calvarial cells. Osteoclast-like cells and tartarate resistant acid phosphatase (TRAP) activities were then quantitated. We examined these cell lines and samples from breast cancer by RT-PCR for the expressions of OPG and RANKL mRNA. Compared to controls, co-culture of MDA-MB-231 cells with stromal or osteoblastic cells induced an increase in number of osteoclasts and TRAP activities. MDA-MB-231 cells alone or breast cancer samples did not express RANKL mRNA. However, co-culture of these cancer cells with stromal or osteoblastic cells induced RANKL mRNA expression and decreased OPG mRNA expression. These experiments demonstrate that direct interactions between breast cancer and stromal or osteoblastic cells induce osteoclastogenesis in vitro through modulating RANKL expression.     

骨保护蛋白和骨保护蛋白配体在脊柱结核病灶骨中的表达

背景：脊柱结核病灶骨组织硬化的病理过程较为复杂,其发生机制尚不明确。 目的：探讨脊柱结核病灶骨不同部位的骨保护蛋白和骨保护蛋白配体的表达。 方法：取脊柱结核患者脊柱结核术中切除的骨质,按照部位分成硬化骨组,亚正常骨组和正常骨组。应用PV-6000二步法免疫组化检测技术测定各组标本中骨保护蛋白和骨保护蛋白配体的平均吸光度及阳性面积比,并进行统计学分析。 结果与结论：脊柱结核硬化骨组骨保护蛋白平均吸光度,阳性面积比以及骨保护蛋白和骨保护蛋白配体表达的比值均显著高于亚正常骨组和正常骨组(P < 0.01或P < 0.05)。结果表明骨保护蛋白和骨保护蛋白配体的表达变化与脊柱结核病灶周围硬化骨的产生有关。     

Increased RANKL expression is related to tumour migration and metastasis of renal cell carcinomas

Receptor activator of NF‐κB ligand (RANKL) and its receptor, receptor activator of NF‐κB (RANK), play a key role in osteoclastogenesis, and osteoprotegerin (OPG) acts as a decoy receptor for RANKL. We investigated the role of the RANKL–RANK–OPG system in renal cell carcinomas (RCCs), which frequently metastasize to bones. Real‐time quantitative PCR revealed that RANKL mRNA expression was higher in clear cell RCCs than in papillary and chromophobe RCCs. Similarly, RANKL protein expression level in clear cell RCCs was higher than that in papillary and chromophobe RCCs, showing positive correlations with the primary tumour stage and distant metastasis. There was no significant association between the expression level of RANK, OPG and histological subtypes of RCC. RANKL and RANK expression was observed in metastatic RCCs in the bone and other organs, suggesting that they play a role in metastasis to the bone and other organs. Recombinant RANKL protein stimulated migration of a clear cell RCC cell line, Caki‐1, in vitro, and this enhanced migration was inhibited by the administration of recombinant OPG protein. Furthermore, multivariate Cox analysis revealed that elevated RANKL and RANK expression with low‐OPG expression was a significant and independent predictor of recurrence, bone metastasis and a poor prognosis. These data suggest that the RANKL–RANK–OPG system is involved not only in the bone metastasis of RCCs but also in metastasis to other organs through the stimulation of cancer cell migration. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

低强度脉冲超声对兔下颌骨骨折愈合中OPG/OPGL及COX-2表达的影响

目的通过观察低强度脉冲超声（low intensity pulsed ultrasound,LIPUS）治疗新西兰大白兔下颌骨骨折后骨保护素（osteoprotegerin,OPG）、骨保护素配体（osteoprotegerin ligand,OPGL）、环氧合酶-2（cyclooxygenase-2,COX-2）蛋白及mRNA的动态变化,探讨LIPUS促进骨折愈合的作用机制。结论健康雄性新西兰大白兔84只,随机分为对照组（42只）和实验组（42只）,建立下颌骨骨折模型,用LIPUS治疗,分别于术后1、3、14、21、28、56d各取相同例数动物（n=6）处死取样。采用免疫组织化学、RT-PCR技术检测OPG、OPGL、COX-2蛋白及mRNA动态变化。结果 LIPUS治疗兔下颌骨骨折后,OPG、OPGL、COX-2蛋白浓度和mRNA及OPGL/OPG比值在不同时间点均有不同程度上调（P〈0.05）,但各指标上调幅度最大（P〈0.01）的时间点不同,且上调幅度无相关性,只有OPG的上调幅度与OPGL比较差异有统计学意义（P〈0.05）。结论 LIPUS能促进兔下颌骨骨折部位OPG、OPGL、COX-2的表达,从而促进骨折愈合。     

Cathepsin K is the principal protease in giant cell tumor of bone

Giant cell tumor (GCT) of bone is a neoplasm of bone characterized by a localized osteolytic lesion. The nature of GCT is an enigma and the cell type(s) and protease(s) responsible for the extensive localized clinicoradiological osteolysis remain unresolved. We evaluated protease expression and cellular distribution of the proteolytic machinery responsible for the osteolysis. mRNA profiles showed that cathepsin K, cathepsin L, and matrix metalloproteinase (MMP)-9 were the preferentially expressed collagenases. Moderate expression was found for MMP-13, MMP-14, and cathepsin S. Specific protease activity assays revealed high cathepsin K activity but showed that MMP-9 was primarily present (98%) as inactive proenzyme. Activities of MMP-13 and MMP-14 were low. Immunohistochemistry revealed a clear spatial distribution: cathepsin K, its associated proton pump V-H(+)-ATPase, and MMP-9 were exclusively expressed in osteoclast-like giant cells, whereas cathepsin L expression was confined to mononuclear cells. To explore a possible role of cathepsin L in osteolysis, GCT-derived, cathepsin L-expressing, mononuclear cells were cultured on dentine disks. No evidence of osteolysis by these cells was found. These results implicate cathepsin K as the principal protease in GCT and suggest that osteoclast-like giant cells are responsible for the osteolysis. Inhibition of cathepsin K or its associated proton-pump may provide new therapeutic opportunities for GCT.     

Telomeric fusion is a major cytogenetic aberration of giant cell tumors of bone

Giant cell tumor of bone (GCT) is regarded as a rare primary bone neoplasm derived from stromal cells, which have the ability to recruit and harbor macrophage and multinucleated osteoclast-like giant cells. Despite being often considered benign, GCT is a problematic neoplasm in that it is aggressive, unpredictable and difficult to treat effectively. Cytogenetically GCT is characterised by a high frequency of telomeric fusion, a process which has been implicated in the production of chromosome instability and tumorigenesis. To extend our knowledge of the significance of telomere association in GCT, the cytogenetics of cell lines derived from spindle-shaped stromal-like mononuclear cells (the tumor cells) of GCT was investigated. Cell lines from three different patients showed telomeric association in all passages. The rate of telomeric association varied from line to line and from passage to passage, but there was no particular pattern to the variations. Many other cytogenetic abnormalities were seen as well as telomeric association, but these were rarely clonal. The nature of most of the other abnormalities seen, such as deleted chromosomes and chromosomes with additional unidentifiable material, was consistent with their being formed as a result of breakage of the dicentric fused chromosomes at a telophase. Chromosomes 13, 14 and 21 were most commonly involved in telomeric fusion. It appears that telomeric association persists in long-term cultures of GCT and is responsible for the accumulation of other associated cytogenetic aberrations. Telomeric reduction and telomerase activity may act as oncogenic events, promoting and sustaining the transformed GCT phenotype.     

Immune function of the decoy receptor osteoprotegerin

Osteoprotegerin (OPG) is a member of the tumor necrosis factor receptor (TNFR) superfamily. OPG has an important function as a protector of bone, demonstrated by the fact that OPG(-/-) mice have severe osteoporosis. OPG acts as a decoy receptor, binding to RANK ligand (RANKL), thus preventing the interaction between receptor activator of NF-kappaB (RANK) and RANKL. This interaction is required for the development of functionally active osteoclasts. Osteoclasts are cells of the monocytic lineage that are responsible for bone resportion. Furthermore, as well as being an important player in the regulation of bone metabolism, OPG also has a role in the regulation of the immune response. Dendritic cells (DCs) express RANK and T cells express RANKL. The ligation of RANK by RANKL can activate both T cells and DCs. Furthermore, both B cells and DCs secrete OPG, and this secretion is regulated by the CD40 receptor. OPG(-/-) mice have B-cell developmental defects. Also, DCs isolated from OPG(-/-) mice more efficiently present antigen in vitro and secrete elevated amounts of inflammatory cytokines when stimulated with bacterial products, or soluble RANKL in vitro. Taken together, this suggests that OPG plays an important role in the immune response regulating the interactions between T cells and DCs.     

OPG/RANKL/RANK系统及其临床应用

人OPG，RANK和RANKL是3个肿瘤坏死因子家族新成员，主要存在于人的骨髓及其他少数组织中。2000年美国骨与矿物质研究协会对其给予了标准化命名，并统称为OPG/RANKL/RANK系统。作为破骨细胞形成、分化和骨吸收调节的关键调节物，OPG/RANKL/RANK系统在骨质疏松、骨硬化病、类风湿性关节炎、骨肿瘤、Paget’s病、牙周炎及正畸牙移动等临床疾病的发病机制及治疗方面均取得了较大进展。     

TRANCE is a TNF family member that regulates dendritic cell and osteoclast function.

Tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE) is a new member of the TNF family emerging as a key regulator of the immune system and of bone development and homeostasis. TRANCE is expressed on activated T cells and activates mature dendritic cells (DC), suggesting that it plays a role in the T cell-DC interaction during an immune response. Furthermore, TRANCE is expressed on osteoblasts stimulated with vitamin D3, dexamethasone, and parathyroid hormone. TRANCE, when expressed on osteoblasts, induces osteoclastogenesis and osteoclast activation, suggesting that it links known calciotropic hormones to bone resorption. TRANCE mediates its effects via the TRANCE-receptor (TRANCE-R/RANK), whereas its activity can be inhibited by the soluble decoy receptor osteoprotegerin/osteoclast inhibitory factor (OPG/OCIF). OPG can be neutralized by another TNF-family member, the TNF-related apoptosis-inducing ligand (TRAIL), suggesting that TRANCE is part of a complex cytokine network that regulates a diverse set of functions. We will discuss the current literature describing TRANCE and its receptors and its role in controlling DC and osteoclast function.     

Giant cell tumor of bone: Frequent actin immunoreactivity in stromal tumor cells

Although giant cell tumor (GCT) of bone is a well-recognized neoplasm with distinctive clinical and histopathological features, the origin of tumor cells, particularly of mononuclear cells, has not yet been established. An immunohistochemicai study was carried out on 11 cases of GCT of bone to examine the cellular natures of stromal mononuclear cells. In all cases, stromal cells were positive for muscle actin (HHF35) or α-smooth muscle actin, and in eight of 11 cases, positivist was intense and extensive. The cell margin of osteoclast-like giant cells (OGC) was stained positively by muscle actin, In addition to Intense and diffuse positive staining of the cytoplasm for KP1 (CD68), whereas α-smooth muscle acting exhibited a negative reaction on the OGC. In conclusion, the tumor cells with muscle actin and α-smooth muscle actin proclivities are not rare but frequently numerous In the GCT of bone; whereas further observation Is necessary to elucidate whether the stromal cells exhibit myoflbroblastlc cell differentiation exactly.     

Deflazacort increases osteoclast formation in mouse bone marrow culture and the ratio of RANKL/OPG mRNA expression in marrow stromal cells.

Information on precise effects of deflazacort on bone cell function, especially osteoclasts, is quite limited. Therefore, the present study was undertaken to test effects of deflazacort on osteoclast-like cell formation in mouse bone marrow cultures and on the regulation of osteoprotegerin (OPG) and its ligand (RANKL) mRNA expressions by RT-PCR in the ST2 marrow stromal cells. TRAP-positive mononuclear cells increased after the treatment of deflazacort at 10(-9) to 10(-7) M alone for 6 days in a dose-dependent manner. Number of TRAP-positive multi-nucleated cells (MNCs) increased significantly with combined treatment of deflazacort at 10(-7) M and 1,25-(OH)2D3 at 10(-9) M compared to that of cultures treated with 1,25-(OH)2D3 alone (p<0.05). Exposure to deflazacort at 10(-7) M in the presence of 1,25-(OH)2D3 at 10(-9) M in the last 3-day culture had greater stimulatory effect on osteoclast-like cell formation than that of the first 3-day culture did. Deflazacort at 10(-10) -10(-6) M downregulated OPG and upregulated RANKL in mRNA levels in a dose-dependent manner. These observations suggest that deflazacort stimulate osteoclast precursor in the absence of 1,25-(OH)2D3 and enhance differentiation of osteoclasts in the presence of 1,25-(OH)2D3. These effects are, in part, thought to be mediated by the regulation of the expression of OPG and RANKL mRNA in marrow stromal cells.     

Bacteria induce osteoclastogenesis via an osteoblast-independent pathway

Bacteria or their products may cause chronic inflammation and subsequent bone loss. This inflammation and bone loss may be associated with significant morbidity in chronic otitis media, periodontitis, endodontic lesions, and loosening of orthopedic implants caused by lipopolysaccharide (LPS)-contaminated implant particles. Currently, it is not clear how bacteria or endotoxin-induced bone resorption occurs and what cell types are involved. Here we report that Porphyromonas gingivalis, a periodontal pathogen, and Escherichia coli LPS induce osteoclastic cell formation from murine leukocytes in the absence of osteoblasts. In contrast, stimulation with parathyroid hormone had no effect. These multinucleated, tartrate-resistant acid phosphatase-positive cells were positive for receptor activator of NF-kappaB (RANK), the receptor for osteoprotegerin ligand (OPGL), also known as RANK ligand (RANKL). Blocking antibodies demonstrated that their formation was dependent upon expression of OPGL and, to a lesser extent, on tumor necrosis factor alpha. Mononuclear cells represented a significant source of OPGL production. In vivo, P. gingivalis injection stimulated OPGL expression in both mononuclear leukocytes and osteoblastic cells. Thus, these findings describe a pathway by which bacteria could enhance osteolysis independently of osteoblasts and suggest that the mix of cells that participate in inflammatory and physiologic bone resorption may be different. This may give insight into new targets of therapeutic intervention.