Functions of RANKL/RANK/OPG in bone modeling and remodeling

The discovery of the RANKL/RANK/OPG system in the mid 1990s for the regulation of bone resorption has led to major advances in our understanding of how bone modeling and remodeling are regulated. It had been known for many years before this discovery that osteoblastic stromal cells regulated osteoclast formation, but it had not been anticipated that they would do this through expression of members of the TNF superfamily: receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG), or that these cytokines and signaling through receptor activator of NF-κB (RANK) would have extensive functions beyond regulation of bone remodeling. RANKL/RANK signaling regulates osteoclast formation, activation and survival in normal bone modeling and remodeling and in a variety of pathologic conditions characterized by increased bone turnover. OPG protects bone from excessive resorption by binding to RANKL and preventing it from binding to RANK. Thus, the relative concentration of RANKL and OPG in bone is a major determinant of bone mass and strength. Here, we review our current understanding of the role of the RANKL/RANK/OPG system in bone modeling and remodeling.     

仙灵骨葆对骨质疏松大鼠OPG/RANK/RANKL 表达的影响

目的:观察仙灵骨葆治疗骨质疏松模型大鼠后,对大鼠体内OPG/RANKL/RANK表达的影响。方法:卵巢摘除法建立SD大鼠骨质疏松模型,设立假手术组、对照组(单纯去卵巢组)、雌激素组(给予17β-雌二醇)和治疗组(给予仙灵骨葆)。术后1周开始给药,给药12周后检测各组大鼠股骨骨密度,ELISA法检测血清中OPG/RANKL含量,RT-PCR检测骨组织中OPG/RANK/RAN-KL mRNA表达,免疫组化检测骨组织中RANK的表达。结果:对照组大鼠骨密度显著低于假手术组;治疗组和雌激素组大鼠O-PG表达显著高于对照组,RANK及RANKL的表达显著低于对照组。结论:采用卵巢摘除法成功建立大鼠骨质疏松模型;仙灵骨葆可促进骨质疏松大鼠OPG的表达,并抑制RANK及RANKL的表达,对骨质疏松模型大鼠有治疗作用。     

OPG、RANK、RANKL的结构、作用机制和在骨疾病中的作用

破骨细胞和成骨细胞分别介导骨的吸收过程和合成过程,而OPG、RANK、RANKL在调节二者的比例中发挥非常重要的作用.RANKL与RANK结合后可能通过三种途径:JNK途径、NF-κB途径和蛋白激酶B途径参与破骨细胞的分化,促进骨质的吸收;RANKL与OPG结合后能阻断RANKL与RANK的结合,由于缺乏RANKL-RANK产生的转录活化信号,破骨细胞分化成熟发生障碍,骨质的吸收受到抑制.OPG、RANK、RANKL同时也是免疫分子,在淋巴细胞、淋巴器官的分化、发育中起重要的作用,骨疾病与免疫系统之间存在着一定的关系.RANMKL/RANK与RANKI/OPG在生物体内保持着一定的比率,如果比率失衡,就会引起各种骨疾病.本篇综述总结了近年来OPG、RANK、RANKL结构、作用的新进展以及它们在骨疾病中的作用.     

Imbalance of RANK,RANKL and OPG expression during tibial fracture repair in diabetic rats

To clarify the mechanisms of altered bone repair in the diabetic state, we investigated RANK, RANKL and OPG expression by immunohistochemistry and RT-PCR in the fracture sites of rats that were either healthy or made diabetic by alloxan. Histomorphometric analysis of the fracture site at 7 days after fracture revealed that diabetic rats (db) have significantly less hard tissue formation at the fracture site, compared to controls. The number of RANK, RANKL and OPG positive cells was decreased in the db group; however, the RANKL/OPG ratio was similar in controls and db at this time. At day 14, numbers of RANKL and OPG positive cells and the mRNA expression for these markers were higher in the control group than in db (P = 0.008). The RANKL/OPG ratio in the db group was greater than in controls. Our results demonstrate an imbalance of RANKL/OPG expression associated with diabetes that may contribute to the delay of fracture repair during the course of diabetes.     

Disturbance of the OPG/RANK/RANKL pathway and systemic inflammation in COPD patients with emphysema and osteoporosis

Background

Osteoporosis is one of the systemic features of COPD. A correlation between the emphysema phenotype of COPD and reduced bone mineral density (BMD) is suggested by some studies, however, the mechanisms underlying this relationship are unclear. Experimental studies indicate that IL-1β, IL-6 and TNF-α may play important roles in the etiology of both osteoporosis and emphysema. The OPG/RANK/RANKL system is an important regulator of bone metabolism, and participates in the development of post-menopausal osteoporosis. Whether the OPG/RANK/RANKL pathway is involved in the pathogenesis of osteoporosis in COPD has not been studied.

Methods

Eighty male patients (current or former smokers) completed a chest CT scan, pulmonary function test, dual x-ray absorptiometry measurements and questionnaires. Among these subjects, thirty patients with normal BMD and thirty patients with low BMD were selected randomly for measurement of IL-1β, IL-6, TNF-α (flow cytometry) and OPG/RANK/RANKL (ELISA). Twenty age-matched healthy volunteers were recruited as controls.

Results

Among these eighty patients, thirty-six had normal BMD and forty-four had low BMD. Age, BMI and CAT score showed significant differences between these two COPD groups (p < 0.05). The low-attenuation area (LAA%) in the lungs of COPD patients was negatively correlated with lumbar vertebral BMD (r = 0.741; p < 0.0001). Forward logistic regression analysis showed that only LAA% (p = 0.005) and BMI (p = 0.009) were selected as explanatory variables. The level of IL-1β was significantly higher in the COPD patients as compared to the normal controls (p < 0.05), but the difference between the two COPD groups did not reach significance. The levels of IL-6 and TNF-α among the three groups were significantly different (p < 0.05). The level of RANKL and the RANKL/OPG ratio were significantly higher in COPD patients with low BMD compared to those with normal BMD and the normal controls (p < 0.05), and correlated negatively with lumbar vertebral BMD, but positively with LAA%.

Conclusions

Radiographic emphysema is correlated with low BMD in current and former smokers with COPD. IL-1β, IL-6, TNF-α, and the osteoporosis-related protein system OPG/RANK/RANKL may have some synergetic effects on emphysema and bone loss in COPD.     

OPG/RANK/RANKL系统与骨质疏松研究最新进展

骨质疏松是严重威胁中老年人健康的骨科常见病,OPG/RANK/RANKL是参与调节骨重建的最重要的分子系统之一,与骨疾病相关的骨质疏松有密切联系,并已成为药物设计的新靶点.因此,对该系统的深入研究将为骨生理、病理机制阐明及骨疾病防治带来积极影响.     

Assessment and clinical implications of RANK/RANKL/OPG pathway as markers of bone tumor progression in patients with NET harboring bone metastases

Abstract

Introduction: The impact on the survival of bone metastases (BM) in patients with neuroendocrine tumor (NET) is a matter of debate. BM have a key role in causing symptoms and in decreasing patients’ quality of life. Although the mechanisms of the development of BM are not completely clear, it is now well understood that the Receptor Activator of Nuclear factor Kappa-B-/Ligand (RANK/RANKL)/osteoprotegerin (OPG) pathway plays a relevant role.

Aim: To characterize the RANK/RANKL/OPG pathway in patients affected with NET.

Patients and methods: Two cohorts of 15 patients each were enrolled in the study; one cohort was affected with NET without BM and the second cohort was affected with NET with BM. The serum RANK/RANKL/OPG pathway was assessed in both the groups.

Results: Serum OPG levels and RANKL/OPG ratio were lower and higher, respectively, in NET patients harboring BM than in those without BM. During the ROC analysis, a cut-off value of 1071?pg/ml for OPG and 0.62 for RANKL/OPG ratio were able to significantly distinguish between the two groups.

Conclusions: This study indicates that RANK/RANKL/OPG pathway is imbalanced in patients with NET harboring BM. Specific alterations of this pathway could predict an early development of BM.     

Osteoprotegerin (OPG) and RANKL expression and distribution in developing human craniomandibular joint

During embryogenesis the bone tissue of craniomandibular joint (CMJ) is formed through two pathways: intramembranous ossification and endochondral ossification. The development process is under the control of regulatory factors.The osteoprotegerin (OPG) and the receptor activator of nuclear factor (NF)-kappaB ligand are key regulators of osteoclastogenesis. The aim of this study is the localization of OPG and RANKL mRNA and protein in the foetal CMJ by immunohistochemistry (IHC) and in situ hybridization (ISH). The main results were: OPG and RANKL mRNA and protein were co-localized in the same cell types; OPG and RANKL were specially immunolocated in osteogenic cells; immunolabeling was often seen in the nucleus and cytoplasm of otherwise negative hypertrophic chondrocytes; IHC and ISH labeling decreased from proliferative to hypertrophic chondrocytes; early osteocytes showed dual protein expression and some of the mature osteocytes were ISH-negative; periosteal osteoclasts and chondroclasts were mostly stained by IHC and variably labeled by ISH; the new bone matrix and trabecular borders showed intense immunolabeling. The co-expression of OPG and RANKL in the same bone cell types confirms their strictly coupled action in the regulation of bone metabolism in the CMJ development and their extracellular presence in the new bone matrix and trabecular borders suggests a local regulatory role.     

兔膝关节损伤后软骨细胞中OPG与RANKL表达

目的:随着中国经济的高速发展,人民生活水平日益增高。体育运动,高能量机动车造成的膝关节损伤数量不断增加,关节软骨的损伤恢复一直不是很理想。针对其后期的恢复研究做了许多相关的实验。本实验通过对兔膝关节软骨细胞损伤后软骨细胞中OPG(骨保护素)与RANKL(核激活因子受体配体)两种因子在损伤后与正常软骨细胞中的表达的比较,探讨损伤后软骨细胞中OPG和RANKL的表达。方法:25只大耳白兔随机分成5组,每组5只。前4组在10天完成20只兔左后腿膝关节软骨损伤动物模型,最后1组5只做手术对照组,术后处置相同。在术后1周,2周,4周,8周时取膝关节软骨损伤处关节软骨制成标本对照组取正常膝关节软骨制成标本。应用HE染色观察软骨细胞恢复情况及SP免疫组化法检测软骨细胞中OPG与RANKL的表达。在光镜下观察,通过医学图像分析软件(media cybernetics image-proplus6.0)图片分析,测定镜下相对灰度值并计算每个标本的平均灰度值,单因素方差分析法进行数据分析。结果:OPG在膝关节损伤后2-4周表达最高,4-8周有下降趋势。RANKL在膝关节软骨损伤后明显表达并逐步的呈升高趋势。实验组镜下观察表达较强烈,测定镜下平均灰度值与对照组有显著性差异(P0.05)。结论:1.OPG在膝关节软骨损伤后显著性表达。2-4周表达较高,4-8周趋于降低。2.RANKL在膝关节软骨损伤后显著表达,1-8周呈平稳上升趋势。     

不同温度下成骨细胞增殖及其OPG/RANKL mRNA表达的影响

目的:探讨不同温度下对小鼠成骨细胞MC3T3-E1的增殖以及OPG/RANKL表达水平的影响。方法:1.以小鼠成骨细胞MC3T3-E1为体外实验模型,MTT法检检测细胞的增殖情况。2.RT-PCR方法检测MC3T3-E1OPG/RANKL mRNA的表达水平。结果:设定对照组为37℃,高于对照组(38℃-39℃-40℃-41℃-42℃)分别作用于MC3T3-E1细胞1小时/天,连续1周,可刺激细胞增殖,OD值显著增加(P<0.05)。同时可增加OPG mRNA表达,降低RANKL mRNA表达,呈温度梯度依赖性。结论:热刺激促进MC3T3-E1细胞增殖,同时通过调节OPG/RANKL mRNA的表达,直接促进骨形成,抑制骨吸收。     

Involvement of integrin up‐regulation in RANKL/RANK pathway of chondrosarcomas migration

Invasion of tumor cells is the primary cause of therapeutic failure in malignant chondrosarcomas treatment. Receptor activator of nuclear factor‐κB ligand (RANKL) and its receptor, RANK, play a key roles in osteoclastogenesis and tumor metastasis. We found that the RANKL and RANK expression in human chondrosarcoma tissues was higher than that in normal cartilage. We also found that RANKL directed the migration and increased cell surface expression of β1 integrin in human chondrosarcoma cells (JJ012 cells). Pretreatment of JJ012 cells with MAPK kinase (MEK) inhibitors, PD98059 or U0126, inhibited the RANKL‐induced migration and integrin expression. Stimulation of cells with RANKL increased the phosphorylation of MEK and extracellular signal‐regulating kinase (ERK). In addition, NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) also inhibited RANKL‐induced cells migration and integrin up‐regulation. Taken together, these results suggest that the RANKL acts through MEK/ERK, which in turn activates IKKα/β and NF‐κB, resulting in the activation of β1 integrin and contributing to the migration of human chondrosarcoma cells. J. Cell. Biochem. 111: 138–147, 2010. © 2010 Wiley‐Liss, Inc.     

Expression of RANKL/OPG during bone remodeling in vivo

The interaction between receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) plays a dominant role in osteoclastogenesis. As both proteins are produced by osteoblast lineage cells, they are considered to represent a key link between bone formation and resorption. In this study, we investigated the expression of RANKL and OPG during bone remodeling in vivo to determine the relationship between osteoclastogenic stimulation and osteoblastic differentiation.Total RNA was prepared from rat femurs after marrow ablation on days 0, 3, 6, and 9. The temporal activation patterns of osteoblast-related genes (procollagen α1 (I), alkaline phosphatase, osteopontin, and osteocalcin) were examined by Northern blot analysis. An appreciable increase in the expression of these osteoblast markers was observed on day 3. The peak increase in gene expression was observed on day 6 followed by a slight reduction by day 9. Real-time PCR analysis showed that the OPG mRNA expression was markedly upregulated on day 6 and slightly decreased on day 9. In contrast, RANKL mRNA expression was increased by more than 20-fold on day 9. The RANKL/OPG ratio, an index of osteoclastogenic stimulation, peaked on day 9. Histological analysis showed that RANKL and OPG immunoreactivity were predominantly associated with bone marrow cells. The expression of bone formation markers was activated in the bone formation phase, followed by the stimulation of RANKL/OPG expression in the bone resorption phase, which confirmed that these molecules are key factors linking bone formation to resorption during bone remodeling.     

胰岛素对2型糖尿病骨质疏松大鼠血清与骨OPG、RANKL表达的影响

目的:探讨胰岛素对2型糖尿病骨质疏松大鼠血清及骨OPG(osteoprotegerin)、RANKL(OPG receptor activator nuclear factork B)表达水平的影响。方法:以高脂高糖饲料喂养4周同时饮用3%果糖水导致胰岛素抵抗小鼠,再以小剂量链脲佐菌素(30mg/kg)腹腔注射1次,2周后诱导建立2型糖尿病小鼠模型。对照组动物则给予正常饲料及饮用水进行喂养。模型建立成功后,对模型2组大鼠进行胰岛素治疗,分别采用OPG和RANKLelisa试剂盒对正常动物模型和糖尿病动物模型血清和骨组织中OPG,RANKL含量进行比较分析,采用血糖分析仪对不同组动物的血糖进行比较分析,采用骨密度分析仪对动物的骨密度进行分析,了解高血糖对于骨密度及血清,骨组织中OPG,RANKL含量的影响以及胰岛素对高血糖骨质疏松造成的结果的影响。结果:相较于正常组大鼠,模型组大鼠血清及髂骨中OPG、血糖、糖化血红蛋白、髂骨密度表达显著下调(P0.05),而RANKL表达显著上调(P0.05),胰岛素处理的模型大鼠血清及骨中OPG含量较模型组大鼠显著升高,血清及骨组织中RANKL表达显著下调(P0.05)。结论:胰岛素能够显著降低2型糖尿病骨质疏松大鼠血清及骨组织中RANKL的表达,显著上调OPG的表达。     

吸烟对种植体周围炎患者龈下菌群、龈沟液炎症因子和RANKL/OPG比值的影响

摘要 目的：探讨吸烟对种植体周围炎患者龈下菌群分布、龈沟液炎症因子白细胞介素-4（IL-4）、白细胞介素-5（IL-5）、白细胞介素-6（IL-6）、白细胞介素-8（IL-8）、白细胞介素-17（IL-17）和核因子-κB受体活化因子配体/骨保护素（RANKL/OPG）比值的影响。方法：选择2019年3月至2022年3月首都医科大学附属北京友谊医院口腔科收治的种植体周围炎患者99例（共151颗种植体）,根据是否吸烟分为吸烟组（45例,68颗种植体）和非吸烟组（54例,83颗种植体）,比较两组患者种植体牙周改良菌斑指数（mPLI）、牙龈出血指数（GBI）、种植体周围探诊深度（PPD）,采集两组龈下菌斑进行细胞培养并进行菌种鉴定,分析两组龈下菌群分布情况,比较两组龈沟液中IL-4、IL-5、IL-6、IL-8、IL-17、RANKL/OPG比值。结果：吸烟组mPLI、GBI、PPD高于不吸烟组（P＜0.05）。吸烟组厌氧菌检出率高于非吸烟组（P＜0.05）,有益菌检出率低于非吸烟组（P＜0.05）,两组需氧菌检出率比较差异无统计学意义（P＞0.05）。吸烟组龈沟液IL-4、IL-5、IL-6、IL-8、IL-17水平均高于非吸烟组（P＜0.05）。吸烟组龈沟液RANKL水平、RANKL/OPG比值高于非吸烟组（P＜0.05）,OPG水平低于非吸烟组（P＜0.05）。结论：吸烟可导致种植体周围炎患者龈下厌氧菌增加,加重炎症反应,增加牙槽骨吸收风险。     

Leptin effect on RANKL and OPG expression in MC3T3-E1 osteoblasts

Recent studies have suggested that leptin hormone may play a pivotal role on bone remodeling through a direct effect by modulating positively the OPG/RANKL balance. Here, we investigate the effect of leptin hormone on RANKL and OPG expression in MC3T3-E1 osteoblasts using RT-PCR and ELISA measurements. We have at first identified the expression of Ob-Rb and Ob-Ra leptin receptor isoforms in MC3T3-E1 and observed that these cells respond to mrleptin treatments. We then investigated the effect of mrleptin on RANKL and OPG expression. We show that mrleptin dose-dependently regulated the expression of RANKL mRNA with complete inhibition observed at concentrations higher than 12 ng/ml. This effect was confirmed with sRANKL protein measurements. However, the exposure of MC3T3-E1 to mrleptin had no effect on OPG mRNA. Taken together, these results suggest that leptin modulates positively OPG/RANKL balance by inhibiting the expression of RANKL gene.     

A novel gene STYK1/NOK is upregulated in estrogen receptor-alpha negative estrogen receptor-beta positive breast cancer cells following estrogen treatment

The human STYK1/NOK protein is approximately 30–35% similar to mouse fibroblast growth factor receptor 3 and a kinase homologue in D. melanogaster in the tyrosine protein kinase region. STYK1/NOK was identified as being up regulated in MDA-MB-231, an estrogen receptor-alpha negative breast cancer cell line, following 12 h of estrogen treatment at 1 × 10⁻⁹ M. On further investigation of STYK1/NOK in estrogen treated cell line MDA-MB-231, STYK1/NOK was up regulated at 6 h post treatment when compared to untreated cells. We also investigated the expression levels of STYK1/NOK in other breast cancer cell lines MCF-7, MDA-MB-231, BT-549, and MDA-MB-435S using QRT-PCR. In addition, the analysis of message accumulation was increased with other synthetic estrogen response modifiers. We propose that the regulation of STYK1/NOK is achieved independent of ERα and suggests further investigation to the relevance of this kinase in breast cancer progression.     

RANK ligand expression in heat shock factor-2 deficient mouse bone marrow stromal/preosteoblast cells

Heat Shock Proteins (HSP) are molecular chaperones activated upon cellular stress/stimuli. HSP gene expression is regulated by Heat Shock Factors (HSF). We have recently demonstrated a functional role for heat shock factor-2 (HSF-2) in fibroblast growth factor-2 (FGF-2)-induced RANK ligand (RANKL), a critical osteoclastogenic factor expression on stromal/preosteoblast cells. In the present study, we show that FGF-2 treatment did not induce RANKL expression in HSF-2-/-stromal/preosteoblast cells. Interestingly, HSF-2 deficiency resulted in rapid induction of alkaline phosphatase (ALP) activity and osteocalcin mRNA expression in these cells. Furthermore, FGF-2 did not induce osteoclast formation in co-culture of normal mouse spleen cells and HSF-2-/-stromal/preosteoblast cells. Electron microscopy analysis demonstrated that osteoclasts from HSF-2-/-mice have poorly developed ruffled borders. These data further confirm that HSF-2 plays an important role in FGF-2-induced RANKL expression in stromal/preosteoblast cells. HSF-2 deficiency has pleotropic effects on gene expression during osteoblast differentiation and osteoclastogenesis in the bone microenvironment. Novel therapeutic agents that modulate HSF-2 activation may have therapeutic utility against increased levels of FGF-2 and bone destruction associated with pathologic conditions.     

Puerarin concurrently stimulates osteoprotegerin and inhibits receptor activator of NF-κB ligand (RANKL) and interleukin-6 production in human osteoblastic MG-63 cells

Puerarin, a daidzein-8-C-glucoside, is the major isoflavone glycoside found in the Chinese herb radix of Pueraria lobata (Willd.) Ohwi, and has received increasing attention because of its possible role in the prevention of osteoporosis. In our previous studies, puerarin reduced the bone resorption of osteoclasts and promoted long bone growth in fetal mouse in vitro. Further study confirmed that puerarin stimulated proliferation and differentiation of osteoblasts in rat. However, the mechanisms underlying its actions on human bone cells have remained largely unknown. Here we show that puerarin concurrently stimulates osteoprotegerin (OPG) and inhibits receptor activator of nuclear factor-κB ligand (RANKL) and Interleukin-6 (IL-6) production by human osteoblastic MG-63 cells containing two estrogen receptor (ER) isotypes. Treatment with the ER antagonist ICI 182,780 abrogates the above actions of puerarin on osteoblast-derived cells. Using small interfering double-stranded RNAs technology, we further demonstrate that the effects of puerarin on OPG and RANKL expression are mediated by both ERα and ERβ but those on IL-6 production primarily by ERα. Moreover, we demonstrate that puerarin may promote activation of the classic estrogen response element (ERE) pathway through increasing ERα, ERβ and steroid hormone receptor coactivator (SRC)-1 expression. Therefore, puerarin will be a promising agent that prevents or retards osteoporosis.