Effect of osteoprotegerin in combination with interleukin-6 on inhibition of osteoclast differentiation

Objective： To observe the effect of recombinant interleukin-6 （IL-6） and osteoprotegerin （OPG） on inhibiting bone absorption induced by receptor activa- tor for nuclear factor- rB ligand （RANKL） in murine osteo- clast precursor cells （OCPs） model. Methods： RAW 264.7 cells were solely treated with 50 ng/ml RANKL for 1 day, and then they were divided into three groups： RANKL （control group）, RANKL＋IL-6 （IL-6 group） and RANKL＋IL-6＋OPG （combination group）. These cells were harvested and investigated by means of HE stain- ing under light microscope after consecutive 9 days. Furthermore, staining tartrate-resistant acid phosphatase （TRAP）-positive multinucleated cells were detected by in- verted phase contrast microscope. The absorption pits of bone slices were observed under scanning electron microscope. Results： The number of mature osteoclast cells in control group was more than that in IL-6 alone or IL-6 com- bined with OPG group （P〈0.05）. Interestingly, this experi- ment has also demonstrated that there was a large number of TRAP-positive multinucleated osteoclasts （more than 3 nuclei） and several bone absorption formation in the con- trol group, whereas the outcome was completely different in both IL-6 group and IL-6＋OPG group （P〈0.05）. Conclusion： IL-6 can suppress the differentiation of mature osteoclasts as directly adding it into the RAW 264.7 cells induced by 50 ng/ml RANKL, and further the effect of osteolysis is remarkably reduced. When treatment with IL- 6 combined with OPG, a more effective strategy for the treat- ment of osteoporosis is reached.     

异丙酚对脂多糖诱导的小鼠巨噬细胞表达释放高迁移率族蛋白1的抑制作用

Objective To explore the inhibition effect of propofol on HMGB1 expression and releasing from mouse macrophage cell RAW264.7 induced by LPS. Methods Detect the time course of HMGB1 expression of RAW264.7 cells using RT-PCR and western blot. Then cells were divided into three groups: control, 250μg/L LPS stimulated, 250μg/L LPS stimulated simultaneously with 50μmol/L propofol. HMGB1 expression and releasing were detected using RT-PCR and western blot when cell were stimulated until the time the HMGB1 expression got to the highest level. Results 16 h after stimulated by LPS, the HMGB1 expression got to the highest level. When LPS stimulated with 50μmol/L propofol, the expression and releasing of HMGB1 by RAW264.7 were inhibited (P<0.05);16 h after stimulated by LPS, the HMGB1 mRNA expression got to the highest level,When LPS stimulated with 50μmol/L propofol, the expression and releasing of HMGB1 mRNA by RAW264.7 were inhibited Significantly,in each group ,the changes of HMGB1 Protein are the same to that of HMGB1 mRNA. Conclusion Propofol can inhibit the expression and releasing of HMGB1 by RAW264.7, which may be play a role in protect of sepsis by LPS.     

异丙酚对脂多糖诱导的小鼠巨噬细胞表达释放高迁移率族蛋白1的抑制作用

Objective To explore the inhibition effect of propofol on HMGB1 expression and releasing from mouse macrophage cell RAW264.7 induced by LPS. Methods Detect the time course of HMGB1 expression of RAW264.7 cells using RT-PCR and western blot. Then cells were divided into three groups: control, 250μg/L LPS stimulated, 250μg/L LPS stimulated simultaneously with 50μmol/L propofol. HMGB1 expression and releasing were detected using RT-PCR and western blot when cell were stimulated until the time the HMGB1 expression got to the highest level. Results 16 h after stimulated by LPS, the HMGB1 expression got to the highest level. When LPS stimulated with 50μmol/L propofol, the expression and releasing of HMGB1 by RAW264.7 were inhibited (P<0.05);16 h after stimulated by LPS, the HMGB1 mRNA expression got to the highest level,When LPS stimulated with 50μmol/L propofol, the expression and releasing of HMGB1 mRNA by RAW264.7 were inhibited Significantly,in each group ,the changes of HMGB1 Protein are the same to that of HMGB1 mRNA. Conclusion Propofol can inhibit the expression and releasing of HMGB1 by RAW264.7, which may be play a role in protect of sepsis by LPS.     

细胞外信号调节蛋白激酶介导醛固酮诱导的肾小球系膜细胞增殖

Objective To determine the role of extracellular signal-regulated kinases (ERK1/2) in aldosterone-induced rat mesangial cells (RMCs) proliferation. Methods RMCs were obtained from intact glomeruli of 4- to 6-week-old Sprague-Dawley rats and characterized according to published methods. RMCs between passages 5 and passages 10 were used. Protein levels of mineralocorticoid receptor(MR) in RMCs were analyzed by Western blotting. The cells were divided into the following groups: control group, PD98059(10 ?滋mol/L) group, eplerenone (1 ?滋mol/L) group, aldosterone (100 nmol/L) group, aldosterone (100 nmol/L) ＋PD98059 (10 ?滋mol/L) group, aldosterone(100 nmol/L)＋eplerenone (1 ?滋mol/L) group. ERK1/2 activity was measured by Western blotting. Cell proliferation of RMCs was evaluated by [3H]-thymidine uptake measurements. Results MR protein expression in RMCs was confirmed by Western blotting. Aldosterone activated ERK1/2, and the maximal ERK1/2 activation induced by aldosterone was at a concentration of 100 nmol/L. Aldosterone (100 nmol/L)-induced activation of ERK1/2 peaked at 10 minutes (P<0.05). Pretreatment with a selective MR antagonist eplerenone (1 ?滋mol/L) significantly attenuated aldosterone-induced ERK1/2 phosphorylation. Aldosterone (100 nmol/L) treatment for 30 hours increased [3H]-thymidine incorporation of RMCs (135%±8% of controls, P<0.05). Cellular proliferation induced by aldosterone could be prevented by pretreatment with eplerenone or an ERK (MEK) inhibitor PD988059. Conclusion Aldosterone induces RMCs proliferation through MR and ERK1/2 activation, which may contribute to the pathogenesis of glomerular mesangial injury.     

塞来昔布对钛颗粒诱导破骨细胞活化的抑制作用

目的 观察环氧合酶-2(COX-2)选择性抑制剂-塞来昔布(Cel)对钛颗粒(Ti)诱导破骨细胞(OC)活化的影响.方法 实验分为6组:空白组、Ti组、核因子(NF)-κB受体活化因子配体(RANKL)组、R+Ti组、Cel组和前列腺素E2(PGE2)组.采用噻唑蓝(MTT)法检测0.lg/L Ti和不同浓度Cel(10、50、100、200、500μg/L)处理小鼠单核/巨噬细胞株RAW264.7后12、24、48、72 h细胞增殖活性;以抗酒石酸酸性磷酸酶(TRAP)染色检测成熟OC,以逆转录-聚合酶链反应(RT-PCR)检测COX-2、核因子(NF)-κB受体活化因子(RANK)、TRAP、活化T细胞核因子c1(NFATc1)和组织蛋白酶K(CPK)基因mRNA含量,Western blot分析COX-2蛋白表达变化,酶联免疫吸附试验(ELISA)检测PGE2表达水平.结果 MTT结果表明Ti和(或)Cel(≤500 μg/L)对RAW264.7细胞增殖能力无影响.Western blot及RT-PCR结果表明COX-2表达峰值出现在RANKL和Ti作用后2h,24 h后COX-2表达量与空白组比较差异无统计学意义(P＞0.05).ELISA结果显示,RANKL和Ti加入后12h,PGE2的含量为3.912±0.231,与Cel预处理组(1.488±0.093)比较,差异有统计学意义(P＜0.05).TRAP染色,RANKL组、R+Ti组、Cel后处理组和PGE2组均可见大量紫红色多核细胞;Ti组和Cel预处理组阳性细胞较少,统计分析结果表明Cel≥100 μg/L时,TRAP阳性细胞数量与RANKL组比较差异有统计学意义(P＜0.05).RT-PCR结果显示Cel组(100 μg/L)RANK、TRAP、NFATcl和CPK基因mRNA含量分别为4.980 ±0.180、3.128±0.143、6.166±0.223和4.922±0.163,与R +Ti组和PGE2组之间的差异有统计学意义(P＜0.05).结论 预先给予选择性COX-2抑制剂Cel能减少PGE2分泌,抑制RANK的表达,阻断Ti诱导的OC活化.     

二甲双胍对破骨细胞体外分化的影响

目的 探讨二甲双胍对破骨细胞体外分化的影响及其可能机制.方法 采用RANKL诱导鼠巨噬细胞系Raw264.7细胞破骨分化模型,给予不同浓度的二甲双胍(400 μmol/L、800 μmol/L和1000μmol/L)和雷帕霉素(100 hmol/L)处理后,通过抗酒石酸酸性磷酸酶(tartrate-resistant Acid Phosphatase,TRAP)染色和破骨细胞骨架结构荧光染色观察破骨细胞数量,骨吸收培养板观察骨陷窝面积,RT-PCR技术检测破骨细胞特异性基因TRAP、组织蛋白酶K、降钙素受体和金属基质蛋白酶-9的表达,ELISA法检测肿瘤坏死因子-α(tumor necrosis factor,TNF-α)表达水平,Western-b1ot检测c-Fos蛋白以及哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin complex l,mTORC1)信号通路下游底物S6K1 Thr389、S6 Ser235/236、4EBP1 Thr37/46的表达及磷酸化水平.结果 二甲双胍和雷帕霉素均可使RANKL诱导的破骨细胞数量减少,抑制破骨细胞特异性基因的表达、抑制TNF-α、c-Fos蛋白以及mTORC1信号通路下游底物S6K1 Thr389、S6 Ser235/236、4E-BP1 Thr37/46的磷酸化,且二甲双胍的抑制作用具有浓度依赖性.结论 二甲双胍可抑制RANKL诱导的破骨前体细胞分化,其机制可能与抑制TNF-α和c-Fos蛋白的生成,以及抑制mTORC1信号通路激活有关.

Abstract：

Objective To investigate the effects of mefformin on the differentiation of osteoclastas well as relative mechanism.Methods Raw264.7 cells from the murine macrophage cell line was used.Receptor activator of NF-κB ligand (RANKL) was used to stimulate osteoclast differentiation from Raw264.7 cells.Osteoclast differentiation was assessed by tartrate-resistant acid phosphatase (TRAP) and actin fluorescence staining and counting the TRAP-positive cells after exposure to different concentrations of mefformin (0 μmol/L,400 μmol/L,800 μmol/L and 1000 μmol/L) or rapamicin (100 nmol/L) in the presence of 50 ng/ml RANKL for 5 days.Bone-resorbing activity was evaluated by BD BioCoatTM OsteologicTM Bone Cell Culture System.The expression of osteoclast-specific genes like TRAP,capthesin K,calcitonin receptor (CTR) and matrix metalloproteinase (MMP-9) was evaluated by RT-PCR.The expression of tumor necrosis factor-α(TNF-ct) S6K1Thr389,S6 Ser235/236,4E-BP1Thr37/46 and c-Fos protein was evaluated by ELISA kit and Western blot analysis,respectively.Results Mefformin dose-dependently inhibited RANKL-stimulated osteoclasts differentiation in Raw264.7 cell culture,as manifested by decrease of TRAP-positive multinucleated cells and pit erosion area,down-regulation of TRAP,cathepsin K,CTR and MMP-9 mRNA and reduction of TNF-α and c-Fos protein expression.Further study revealed that RANKL activated mTOR complex 1(mTORC1) signaling,while mefformin impaired RANKL-stimulated mTORC1 signaling.Rapamycin,an mTORCl-specific inhibitor and immunosuppressive macrolides could also prevent RANKL-induced osteoclast differentiation and bone resorption in vitro.Conclusion Mefformin inhibits osteoclastogenesis in vitro,which may due to reduction of TNF-α and c-Fos protein expression,and mTORC1 signaling is involved in this process.     

高糖及TNF-α对RAW264.7细胞向破骨细胞分化的影响

【摘要】 目的 观察高糖及TNF-α的培养条件对RAW264.7细胞向破骨细胞诱导分化的影响。方法 在正常、高糖(30 mmol/L)及TNF-α(10 μmol/L)条件下培养RAW264.7细胞后,加入浓度为100 ng/mL的细胞核转录因子κB受体激活物的配体(receptor activator of NF-κB ligand, RANKL)为诱导剂,诱导RAW264.7向破骨细胞分化;诱导9天后,抗酒石酸酸性磷酸酶(TRAP)染色,比较各组TRAP+细胞数,RT-PCR及Western Blot检测各组破骨细胞标志基因CTR和MMP-9的表达。结果 不同的培养条件下RANKL均能诱导RAW264.7分化为成熟的破骨细胞,其中TNF-α环境中RAW264.7形成的TRAP+阳性细胞数、CTR和MMP-9的表达最高,而在高糖环境下最低。结论 TNF-α可以促进RAW264.7向破骨细胞分化,而高糖对这个过程可能是抑制作用,这一现象符合Ⅰ型和Ⅱ糖尿病患者骨质破坏的表现;高糖及TNF-α的培养条件下RANKL对RAW264.7的作用可模拟糖尿病足病变微环境中OC的诱导分化的过程。     

NF-kappaB p50 and p52 expression is not required for RANK-expressing osteoclast progenitor formation but is essential for RANK- and cytokine-mediated osteoclastogenesis.

Expression of RANKL by stromal cells and of RANK and both NF-kappaB p50 and p52 by osteoclast precursors is essential for osteoclast formation. To examine further the role of RANKL, RANK, and NF-KB signaling in this process, we used NF-kappaB p50-/- ;p52-/- double knockout (dKO) and wild-type (WT) mice. Osteoclasts formed in cocultures of WT osteoblasts with splenocytes from WT mice but not from dKO mice, a finding unchanged by addition of RANKL and macrophage colony-stimulating factor (M-CSF). NF-kappaB dKO splenocytes formed more colony-forming unit granulocyte macrophage (CFU-GM) colonies than WT cells, but no osteoclasts were formed from dKO CFU-GM colonies. RANKL increased the number of CFU-GM colonies twofold in WT cultures but not in dKO cultures. Fluorescence-activated cell sorting (FACS) analysis of splenocytes from NF-kappaB dKO mice revealed a two-to threefold increase in the percentage of CD11b (Mac-1) and RANK double-positive cells compared with WT controls. Treatment of NF-kappaB dKO splenocytes with interleukin (IL)-1, TNF-alpha, M-CSF, GM-CSF, and IL-6 plus soluble IL-6 receptor did not rescue the osteoclast defect. No increase in apoptosis was observed in cells of the osteoclast lineage in NF-kappaB dKO or p50-/-;p52+/- (3/4KO) mice. Thus, NF-kappaB p50 and p52 expression is not required for formation of RANK-expressing osteoclast progenitors but is essential for RANK-expressing osteoclast precursors to differentiate into TRAP+ osteoclasts in response to RANKL and other osteoclastogenic cytokines.     

低分子量褐藻糖胶对破骨细胞凋亡的影响

目的探讨低分子量褐藻糖胶(LMWF)对小鼠单核细胞RAW264.7诱导成熟破骨细胞凋亡的影响。方法通过100ng/m L RANKL诱导RAW264.7细胞株分化为破骨细胞,经TRAP特异性染色和骨吸收陷窝对诱导后的细胞进行鉴定。鉴定成功后,用100 ng/m L RANKL诱导RAW264.7细胞株5 d后,使用含有LMWF的培养基继续培养3 d,通过对TRAP阳性细胞计数和分析骨吸收面积来观察低分子量褐藻糖胶对破骨细胞的抑制和骨吸收功能情况;采用流式细胞术检测LMWF对破骨细胞凋亡的影响,capsase-3活性测试试剂盒检测LMWF对capsase-3活性进行测定;RT-PCR检测LMWF对成熟破骨细胞BAX与BCL-2基因表达的影响。结果单纯采用100 ng/m L的RANKL可成功诱导成熟的、有功能的破骨细胞。LMWF可以明显抑制RANKL诱导成熟破骨细胞的形成以及成熟破骨细胞的骨吸收功能;流式细胞术显示LMWF可增加成熟破骨细胞的早期凋亡率;并且能升高capsase-3的活性;PCR显示LMWF可明显下调破骨细胞凋亡相关的BCL-2和上调BAX基因mRNA表达,降低BCL-2/BAX的比值。结论低分子量褐藻糖胶可抑制破骨细胞的活性与骨吸收能力,促进破骨细胞凋亡,其主要机制是通过下调BCL-2和上调BAX mRNA基因表达实现的。     

盐酸二甲双胍抑制破骨细胞分化的研究

目的探讨盐酸二甲双胍对破骨细胞分化抑制作用的研究。方法核因子-κB受体活化因子配体(RANKL)诱导小鼠骨髓巨噬细胞分化成破骨细胞,在诱导过程中加入不同浓度的盐酸二甲双胍。培养7 d后固定,抗酒石酸酸性磷酸酶染色,计数破骨细胞的数量,骨吸收培养板观察骨陷窝面积,Western blot检测盐酸二甲双胍对ERK磷酸化的影响。结果盐酸二甲双胍能抑制RANKL诱导的破骨细胞的形成,并且能减少骨陷窝面积,以及抑制ERK磷酸化。结论盐酸二甲双胍可抑制破骨细胞分化及功能,其机制可能与抑制ERK磷酸化有关。     

不同浓度地西他滨对破骨细胞分化的影响

目的：探讨不同浓度梯度地西他滨对破骨细胞形成、活性及吸收功能的影响。方法不同浓度地西他滨（0、0.1、0.25和0.5μmol/L）处理单核巨噬（RAW264.7）细胞。通过4’,6?联眯?2?苯基吲哚（4,6?Diamidino?2?phenylindole dihydrochloride, DAPI）染色和微丝绿色荧光探针（F?actin?Trakcer Green）染色后观察F?actin环的形成即破骨细胞轮廓;抗酒石酸酸性磷酸酶检测试剂盒检测细胞上清中的抗酒石酸酸性磷酸酶活性,骨板吸收实验检测破骨细胞的骨吸收能力;Q?PCR实验检测破骨细胞标志基因抗酒石酸酸性磷酸酶（tartrate?resistant acid phosphatase, TRAP）、组织蛋白酶K（cathepsin K, CK）和脊髓基质金属蛋白酶?9（matrix metalloproteinase?9, MMP?9）的mRNA表达。结果不同浓度地西他滨抑制核因子NF?κB配体激活因子（receptor activator of NF?κB ligand, RANKL）诱导RAW264.7细胞形成F?actin环,降低了破骨细胞的TRAP酶活性,抑制了破骨细胞的骨吸收能力,同时也下调了破骨细胞标志基因TRAP、CK和MMP?9的mRNA表达。且随着药物浓度的增高,上述的抑制作用越明显。结论地西他滨抑制破骨细胞形成、活性和骨吸收能力,且这种抑制作用随着药物浓度的增加而逐渐增强。     

重组人骨保护素与重组核因子κb活化因子受体蛋白对破骨前体细胞分化的影响

目的:对比重组人骨保护素(rhOPG-Fc)与重组核因子κb活化因子受体蛋白(rhRANK)对破骨前体细胞分化的影响.方法:采用成骨细胞与破骨前体细胞RAW264.7混合培养,在地塞米松、1,25 (OH) 2VitD3诱导下生成破骨细胞的方法.研究分3组:rhRANK组:10-5 g/L;rhOPG-Fc组:10-5 g/L;空白对照组.作用9d后观察破骨细胞数目和形态,抗酒石酸酸性磷酸酶(TRAP)染色阳性细胞个数,骨磨片吸收陷窝计数.结果:在空白对照组,小鼠成骨细胞与破骨前体细胞RAW264.7混合培养6d后,开始出现多核细胞,9d时可见大量成熟多核细胞,经TRAP染色证实为成熟破骨细胞,而rhRANK组及rhOPG-Fc组TRAP染色阳性多核细胞数较对照组均减少,特别是rhRANK组减少更明显.骨片吸收陷窝计数显示rhRANK组及rhOPG-Fc组较对照组也明显减少,而相对来说,rhRANK组较rhOPG-Fc组更少.结论:rhOPG-Fc与rhRANK均可以有效抑制破骨前体细胞分化成为成熟破骨细胞,且rhRANK较rhOPG-Fc抑制效果更明显.     

Regulation of RANKL‐induced osteoclastogenesis by RING finger protein RNF114

Normal bone remodeling is a continuous process orchestrated by bone‐resorbing osteoclasts and bone‐forming osteoblasts, which an imbalance in bone remodeling results in metabolic bone diseases. RANKL, a member of the TNF cytokine family, functions as a key stimulator for osteoclast differentiation and maturation. Here, we report that RNF114, previously identified as a psoriasis susceptibility gene, plays a regulatory role in the RANKL/RANK/TRAF6 signaling pathway that mediates osteoclastogenesis. Our results demonstrated that RNF114 expression was significantly down‐regulated in mouse osteoclast precursor cells undergoing RANKL‐induced osteoclast differentiation. RNF114 knockout did not affect development or viability of the subpopulation of bone marrow macrophages capable of differentiating into osteoclasts in culture. However, in the presence of RANKL, RNF114 knockout bone marrow macrophages exhibited enhanced cell proliferation and augmented osteoclast differentiation, as shown by an increased expression of mature osteoclast markers, increased osteoclastic TRAP activity and bone resorption. Conversely, ectopic expression of RNF114 inhibited CTSK expression, TRAP activity, and bone resorption in RANKL‐treated pre‐osteoclasts. RNF114 also suppressed RANKL‐activated NFATc1 expression and NFAT‐regulated promoter activity. RNF114 suppressed TRAF6‐, but not TAK1/TAB2‐mediated NF‐κB activation downstream of RANKL/RANK. In particular, TRAF6 protein levels were down‐regulated by RNF114, possibly via K48‐mediated proteasome‐dependent degradation. These data suggested that RNF114's inhibitory effect on RANKL‐stimulated osteoclastogenesis was mediated by blocking RANK/TRAF6/NF‐κB signal transduction. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:159–166, 2018.     

The RANKL/RANK/OPG pathway

Understanding of osteoclast formation and activation has advanced considerably since the discovery of the RANKL/RANK/OPG system in the mid 1990s. Osteoblasts and stromal stem cells express receptor activator of NF-jB ligand (RANKL), which binds to its receptor, RANK, on the surface of osteoclasts and their precursors. This regulates the differentiation of precursors into multinucleated osteoclasts and osteoclast activation and survival both normally and in most pathologic conditions associated with increased bone resorption. Osteoprotegerin (OPG) is secreted by osteoblasts and osteogenic stromal stem cells and protects the skeleton from excessive bone resorption by binding to RANKL and preventing it from interacting with RANK. The RANKL/OPG ratio in bone marrow is thus an important determinant of bone mass in normal and disease states. RANKL/RANK signaling also regulates lymph node formation and mammary gland lactational hyperplasia in mice, and OPG protects large arteries of mice from medial calcification. This article reviews the roles of the RANKL/RANK/OPG system in bone and other tissues.     

Inhibition of osteoclastogenesis by RNA interference targeting RANK

ABSTRACT: BACKGROUND: Osteoclasts and osteoblasts regulate bone resorption and formation to allow bone remodeling and homeostasis. The balance between bone resorption and formation is disturbed by abnormal recruitment of osteoclasts. Osteoclast differentiation is dependent on the receptor activator of nuclear factor NF-kappa B (RANK) ligand (RANKL) as well as the macrophage colony-stimulating factor (M-CSF). The RANKL/RANK system and RANK signaling induce osteoclast formation mediated by various cytokines. The RANK/RANKL pathway has been primarily implicated in metabolic, degenerative and neoplastic bone disorders or osteolysis. The central role of RANK/RANKL interaction in osteoclastogenesis makes RANK an attractive target for potential therapies in treatment of osteolysis. The purpose of this study was to assess the effect of inhibition of RANK expression in mouse bone marrow macrophages on osteoclast differentiation and bone resorption. METHODS: Three pairs of short hairpin RNAs (shRNA) targeting RANK were designed and synthesized. The optimal shRNA was selected among three pairs of shRNAs by RANK expression analyzed by Western blot and Real-time PCR. We investigated suppression of osteoclastogenesis of mouse bone marrow macrophages (BMMs) using the optimal shRNA by targeting RANK. RESULTS: Among the three shRANKs examined, shRANK-3 significantly suppressed [88.3%] the RANK expression (p < 0.01). shRANK-3 also brought about a marked inhibition of osteoclast formation and bone resorption as demonstrated by tartrate--resistant acid phosphatase (TRAP) staining and osteoclast resorption assay. The results of our study show that retrovirus-mediated shRANK-3 suppresses osteoclast differentiation and osteolysis of BMMs. CONCLUSIONS: These findings suggest that retrovirus-mediated shRNA targeting RANK inhibits osteoclast differentiation and osteolysis. It may appear an attractive target for preventing osteolysis in humans with a potential clinical application.