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.     

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。     

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.     

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.     

Dexamethasone influences human clock gene expression in bronchial epithelium and peripheral blood mononuclear cells in vitro

We determined whether human peripheral blood mononuclear cells (PBMCs) could be used to analyze clock genes by studying their mRNA expressions in human bronchial epithelium (BEAS-2B) and PBMCs following stimulation by the glucocorticoid homologue dexamethasone (DEX) in vitro. PBMCs were obtained at 10:00 h from two diurnally active (∼07:00 to 23:00 h) healthy volunteers and were evaluated for hPer1 mRNA expression following DEX stimulation in vitro using real time-PCR analysis. DEX stimulation of human BEAS-2B cells and PBMCs in vitro led to a remarkable increase of hPer1 mRNA. The glucocorticoid rapidly affected the expression of hPer1 mRNA in PBMCs, suggesting that human PBMCs may be a useful surrogate marker for the investigation of drug effects on clock genes.     

Runx-2 is not essential for the vitamin D-regulated expression of RANKL and osteoprotegerin in osteoblastic cells

The differentiation and activity of osteoclasts are positively and negatively controlled by receptor activator of nuclear factor-kappaB ligand (RANKL), which is expressed on the surface of osteoblasts and stromal cells, and its decoy receptor osteoprotegerin (OPG), which is secreted by osteoblasts and stromal cells, respectively. The expression of the genes for RANKL and OPG is regulated by 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)]. Runt-related gene-2 (Runx-2) is essential for osteoblast differentiation and there are several reports that Runx-2 is involved in osteoclast formation. Therefore, to clarify the role of Runx-2 in osteoclastogenesis, we designed a series of experiments using C2 cells and C6 cells, which are derived from calvariae of runx2-deficient mice. Treatment of C2 cells and C6 cells with 1alpha,25(OH)(2)D(3) for 2-4 days increased and decreased the levels of expression of the mRNAs for RANKL and OPG, respectively, and the effects were dose-dependent. However, by day 8, the level of RANKL mRNA had fallen and that of OPG mRNA had risen. Furthermore, C6 cells induced the differentiation of mouse spleen cells into tartrate-resistant acid phosphatase-positive (TRAP-positive) multinucleated cells (osteoclast-like cells) in the presence of 10(-7)M 1alpha,25(OH)(2)D(3). Such formation of osteoclast-like cells was inhibited by exogenous OPG in a dose-dependent manner. Thus, our findings indicate that Runx-2 is not essential for the expression of RANKL and OPG, and the formation of osteoclast-like cells.     

Cytoskeleton of human mononuclear cells as a possible peripheral marker for phenylalanine neurotoxicity in PKU

In this work we tested human mononuclear cells as a peripheral marker to study neurotoxicity of phenylalanine (Phe). Slices of cerebral cortex of rats or human mononuclear cells were incubated with different concentrations of Phe and/or Ala in the presence of ³²P-orthophosphate, the cytoskeletal fraction was extracted, and the radioactivity incorporated into intermediate filament proteins was measured. Our results show that 2 mM Phe as well as 1 mM Ala are effective in increasing the ³²P in vitro incorporation into IFs in both tissues. When cerebral cortex slices or mononuclear cells were incubated with different concentrations of Phe and/or Ala, the effects on the ³²P in vitro incorporation into IF proteins was compatible with an antagonistic mechanism of action of the two amino acids on the enzymes of the phosphorylating system. In addition, these blood cells may be a possible peripheral marker to study neurotoxicity of Phe in patients with PKU.     

Characterization of osteoprotegerin binding to glycosaminoglycans by surface plasmon resonance: role in the interactions with receptor activator of nuclear factor kappaB ligand (RANKL) and RANK

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of nuclear factor kappaB ligand (RANKL), a key inducer of osteoclastogenesis via its receptor RANK. We previously showed that RANK, RANKL, and OPG are able to form a tertiary complex and that OPG must be also considered as a direct effector of osteoclast functions. As OPG contains a heparin-binding domain, the present study investigated the interactions between OPG and glycosaminoglycans (GAGs) by surface plasmon resonance and their involvement in the OPG functions. Kinetic data demonstrated that OPG binds to heparin with a high-affinity (KD: 0.28 nM) and that the pre-incubation of OPG with heparin inhibits in a dose-dependent manner the OPG binding to the complex RANK-RANKL. GAGs from different structure/origin (heparan sulfate, dermatan sulfate, and chondroitin sulfate) exert similar activity on OPG binding. The contribution of the sulfation pattern and the size of the oligosaccharide were determined in this inhibitory mechanism. The results demonstrated that sulfation is essential in the OPG-blocking function of GAGs since a totally desulfated heparin loses its capacity to bind and to block OPG binding to RANKL. Moreover, a decasaccharide is the minimal structure that totally inhibits the OPG binding to the complex RANK-RANKL. Western blot analysis performed in 293 cells surexpressing RANKL revealed that the pre-incubation of OPG with these GAGs strongly inhibits the OPG-induced decrease of membrane RANKL half-life. These data support an essential function of the related glycosaminoglycans heparin and heparan sulfate in the activity of the triad RANK-RANKL-OPG.     

Vitamin C-deficiency stimulates osteoclastogenesis with an increase in RANK expression

The effects of vitamin C (VC) on osteoclastogenesis were studied in vivo using ascorbate-requiring Osteogenic Disorder Shionogi (ODS) rats and in vitro using bone marrow-derived monocyte/macrophage cells (BMMs). The results confirmed previous findings of increases in the number of osteoclasts and in bone resorption at 2 and 3 weeks, but not 1 week, of VC-deficiency in ODS rats. The mRNA and protein levels of receptor activator nuclear factor kappaB (RANK) ligand and osteoprotegerin, and the mRNA level of macrophage-colony stimulating factor (M-CSF) in the proximal tibia of VC-deficient rats did not differ from those in VC-supplemented control rats. However, the mRNA levels of RANK, c-fos and c-jun were significantly increased at as early as 1 week of VC-deficiency. These results suggested that VC-deficiency stimulated osteoclastogenesis by increasing RANK expression. The osteoclastic differentiation of BMMs was suppressed in the presence of VC. The suppressed osteoclastogenesis was associated with decreased levels of RANK, c-fos and c-jun. The pretreatment of BMMs with VC or PD 98059, a specific inhibitor of extracellular signal regulated kinase (ERK)-activating MEK1, decreased the expression of RANK induced by M-CSF. VC inhibited the M-CSF-induced activation of ERK. These results suggested that VC-deficiency increased osteoclastogenesis by increasing RANK expression mediated through the activation of ERK.