Biology of RANK,RANKL, and osteoprotegerin

The discovery of the receptor activator of nuclear factor-kappaB ligand (RANKL)/RANK/osteoprotegerin (OPG) system and its role in the regulation of bone resorption exemplifies how both serendipity and a logic-based approach can identify factors that regulate cell function. Before this discovery in the mid to late 1990s, it had long been recognized that osteoclast formation was regulated by factors expressed by osteoblast/stromal cells, but it had not been anticipated that members of the tumor necrosis factor superfamily of ligands and receptors would be involved or that the factors involved would have extensive functions beyond bone remodeling. RANKL/RANK signaling regulates the formation of multinucleated osteoclasts from their precursors as well as their activation and survival in normal bone remodeling and in a variety of pathologic conditions. OPG protects the skeleton from excessive bone resorption by binding to RANKL and preventing it from binding to its receptor, RANK. Thus, RANKL/OPG ratio is an important determinant of bone mass and skeletal integrity. Genetic studies in mice indicate that RANKL/RANK signaling is also required for lymph node formation and mammary gland lactational hyperplasia, and that OPG also protects arteries from medial calcification. Thus, these tumor necrosis factor superfamily members have important functions outside bone. Although our understanding of the mechanisms whereby they regulate osteoclast formation has advanced rapidly during the past 10 years, many questions remain about their roles in health and disease. Here we review our current understanding of the role of the RANKL/RANK/OPG system in bone and other tissues.     

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.     

RANKL/RANK/OPG: new therapeutic targets in bone tumours and associated osteolysis

The emergence of the molecular triad osteoprotegerin (OPG)/Receptor Activator of NF-kB (RANK)/RANK Ligand (RANKL) has helped elucidate a key signalling pathway between stromal cells and osteoclasts. The interaction between RANK and RANKL plays a critical role in promoting osteoclast differentiation and activation leading to bone resorption. OPG is a soluble decoy receptor for RANKL that blocks osteoclast formation by inhibiting RANKL binding to RANK. The OPG/RANK/RANKL system has been shown to be abnormally regulated in several malignant osteolytic pathologies such as multiple myeloma [MM, where enhanced RANKL expression (directly by tumour cells or indirectly by stromal bone cells or T-lymphocytes)] plays an important role in associated bone destruction. By contrast, production of its endogenous counteracting decoy receptor OPG is either inhibited or too low to compensate for the increase in RANKL production. Therefore, targeting the OPG/RANK/RANKL axis may offer a novel therapeutic approach to malignant osteolytic pathologies. In animal models, OPG or soluble RANK was shown both to control hypercalcaemia of malignancy and the establishment and progression of osteolytic metastases caused by various malignant tumours. To this day, only one phase I study has been performed using a recombinant OPG construct that suppressed bone resorption in patients with multiple myeloma or breast carcinoma with radiologically confirmed bone lesions. RANK-Fc also exhibits promising therapeutic effects, as revealed in animal models of prostate cancer and multiple myeloma. If the animal results translate to similar clinical benefits in humans, using RANK-Fc or OPG may yield novel and potent strategies for treating patients with established or imminent malignant bone diseases and where standard therapeutic regimens have failed.     

Regulation of osteoprotegerin pro- or anti-tumoral activity by bone tumor microenvironment

Tumor development in bone is often associated with fractures, bone loss and bone pain, and improvement is still needed in therapeutic approaches. Bone tumors are related to the existence of a vicious cycle between bone resorption and tumor proliferation in which the molecular triad osteoprotegerin (OPG)/receptor activator of NF-kappaB (RANK)/RANK ligand (RANKL) plays a pivotal role. RANKL, a member of the TNF superfamily, is one of the main inducers of bone resorption. Its soluble receptor OPG represents a promising therapeutic candidate as it prevents bone lesions and inhibits associated tumor growth. However, its therapeutic use in bone tumors remains controversial due to its ability to bind and inhibit another member of the TNF superfamily, TNF related apoptosis inducing ligand (TRAIL), which is a potent inducer of tumor cell apoptosis. Through its heparin binding domain, OPG is also able to bind proteoglycans present in the bone matrix. This paper is an overview of the involvement of the micro-environment, as represented by the balance of RANKL/TRAIL and the presence of proteoglycans in the regulation of OPG biological activity in bone tumors.     

The molecular triad OPG/RANK/RANKL: involvement in the orchestration of pathophysiological bone remodeling

The past decade has seen an explosion in the field of bone biology. The area of bone biology over this period of time has been marked by a number of key discoveries that have opened up entirely new areas for investigation. The recent identification of the receptor activator of nuclear factor κB ligand (RANKL), its cognate receptor RANK, and its decoy receptor osteoprotegerin (OPG) has led to a new molecular perspective on osteoclast biology and bone homeostasis. Specifically, the interaction between RANKL and RANK has been shown to be required for osteoclast differentiation. The third protagonist, OPG, acts as a soluble receptor antagonist for RANKL that prevents it from binding to and activating RANK. Any dysregulation of their respective expression leads to pathological conditions such as bone tumor-associated osteolysis, immune disease, or cardiovascular pathology. In this context, the OPG/RANK/RANKL triad opens novel therapeutic areas in diseases characterized by excessive bone resorption. The present article is an update and extension of an earlier review published by Kwan Tat et al. [Kwan Tat S, Padrines M, Théoleyre S, Heymann D, Fortun Y. IL-6, RANKL, TNF-/IL-1: interrelations in bone resorption pathophysiology. Cytokine Growth Factor Rev 2004;15:49–60].     

Osteoprotegerin and inflammation

RANK, RANKL, and OPG have well established regulatory effects on bone metabolism. RANK is expressed at very high levels on osteoclastic precursors and on mature osteoclasts, and is required for differentiation and activation of the osteoclast. The ligand, RANKL binds to its receptor RANK to induce bone resorption. RANKL is a transmembrane protein expressed in various cells type and particularly on osteoblast and activated T cells. RANKL can be cleaved and the soluble form is active. Osteoprotegerin decoy receptor (OPG), a member of the TNF receptor family expressed by osteoblasts, strongly inhibits bone resorption by binding with high affinity to its ligand RANKL, thereby preventing RANKL from engaging its receptor RANK. This system is regulated by the calciotropic hormones. Conversely, the effects of RANKL, RANK, and OPG on inflammatory processes, most notably on the bone resorption associated with inflammation, remain to be defined. The RANK system seems to play a major role in modulating the immune system. Activated T cells express RANKL messenger RNA, and knock-out mice for RANKL acquire severe immunological abnormalities and osteopetrosis. RANKL secretion by activated T cells can induce osteoclastogenesis. These mechanisms are enhanced by cytokines such as TNF-alpha, IL-1, and IL-17, which promote both inflammation and bone resorption. Conversely, this system is blocked by OPG, IL-4, and IL-10, which inhibit both inflammation and osteoclastogenesis. These data may explain part of the abnormal phenomena in diseases such as rheumatoid arthritis characterized by both inflammation and destruction. Activated T cells within the rheumatoid synovium express RANKL. Synovial cells are capable of differentiating to osteoclast-like cells under some conditions, including culturing with M-CSF and RANKL. This suggests that the bone erosion seen in rheumatoid arthritis may result from RANKL/RANK system activation by activated T cells. This opens up the possibility that OPG may have therapeutic effects mediated by blockade of the RANKL/RANK system.     

IL-6, leukemia inhibitory factor,and oncostatin M stimulate bone resorption and regulate the expression of receptor activator of NF-kappa B ligand,osteoprotegerin, and receptor activator of NF-kappa B in mouse calvariae

IL-6, leukemia inhibitory factor (LIF), and oncostatin M (OSM) are IL-6-type cytokines that stimulate osteoclast formation and function. In the present study, the resorptive effects of these agents and their regulation of receptor activator of NF-kappaB ligand (RANKL), RANK, and osteoprotegerin (OPG) were studied in neonatal mouse calvaria. When tested separately, neither human (h) IL-6 nor the human soluble IL-6R (shIL-6R) stimulated bone resorption, but when hIL-6 and the shIL-6R were combined, significant stimulation of both mineral and matrix release from bone explants was noted. Semiquantitative RT-PCR showed that hIL-6 plus shIL-6R enhanced the expression of RANKL and OPG in calvarial bones, but decreased RANK expression. Human LIF, hOSM, and mouse OSM (mOSM) also stimulated 45Ca release and enhanced the mRNA expression of RANKL and OPG in mouse calvaria, but had no effect on the expression of RANK. In agreement with the RT-PCR analyses, ELISA measurements showed that both hIL-6 plus shIL-6R and mOSM increased RANKL and OPG proteins. 1,25-Dihydroxyvitamin D3 (D3) also increased the RANKL protein level, but decreased the protein level of OPG. OPG inhibited 45Ca release stimulated by RANKL, hIL-6 plus shIL-6R, hLIF, hOSM, mOSM, and D3. An Ab neutralizing mouse gp130 inhibited 45Ca release induced by hIL-6 plus shIL-6R. These experiments demonstrated stimulation of calvarial bone resorption and regulation of mRNA and protein expression of RANKL and OPG by D3 and IL-6 family cytokines as well as regulation of RANK expression in preosteoclasts/osteoclasts of mouse calvaria by D3 and hIL-6 plus shIL-6R.     

OPG/RANKL/RANK系统与骨破坏性疾病

近年来发现的OPG/RANKL/RANK系统在破骨细胞生成中起着至关重要的作用,是骨骼生理研究领域的重大进展。成骨细胞、骨髓基质细胞、激活的T淋巴细胞表达RANKL,与破骨细胞前体细胞或成熟破骨细胞表面上的RANK结合后,促进破骨细胞的分化及骨吸收活性。成骨细胞及骨髓基质细胞分泌表达OPG可与RANKL竞争性结合,从而阻断RANKL与RANK之间的相互作用。体内多种激素或因子通过影响骨髓微环境内的OPG/RANKL比率来调节骨代谢。此外,乳腺上皮细胞表达有RANK,孕期在性激素的诱导下可表达RANKL,OPG/RANKL/RANK系统在孕期乳腺发育以及母体向胎儿的钙转运过程中发挥重要作用。阻断RANKL/RANK通路有望给骨质疏松、类风湿关节炎及癌症骨转移等骨破坏性疾病的治疗开辟新的途径。进一步研究应了解OPG/RANKL/RANK系统与其它信号传导途径的关系,重视骨骼、免疫及内分泌系统之间的相互作用。目前,开发与OPG功能相似或促进其表达的合成药物有可能成为具有良好经济效益和社会效益的产业。     

RANK ligand

RANK ligand (RANKL), a key mediator of bone resorption in normal and pathological states, is expressed as membrane-bound or soluble forms by tissues as diverse as lymph nodes, spleen, thymus and bone-forming cells. In normal bone turnover and in bone metastasis, RANKL stimulates the formation and activity of bone-removing cells, osteoclasts, by binding to its cognate receptor, RANK, on osteoclasts and their progenitors; these processes are disrupted by binding of RANKL to osteoprotegerin (OPG), a soluble decoy receptor. Whilst no mutations in the RANKL gene have yet been identified in human disease, mutations that result in enhanced RANK signalling through inactivation of OPG or activation of RANK are associated with Juvenile Paget's disease and familial expansile osteolysis, respectively. This review focuses on the central role of RANKL in bone resorption and on the therapeutic targeting of RANKL in osteoporosis, humoral hypercalcaemia of malignancy and bone metastasis.     

Crystal structure of human RANKL complexed with its decoy receptor osteoprotegerin

Receptor activator of NF-κB ligand (RANKL), its signaling receptor RANK, and its decoy receptor osteoprotegerin (OPG) constitute a molecular triad that is critical in regulating bone remodeling, and also plays multiple roles in the immune system. OPG binds RANKL directly to block its interaction with RANK. In this article, we report the 2.7-? crystal structure of human RANKL trimer in complex with the N-terminal fragment of human OPG containing four cysteine-rich TNFR homologous domains (OPG-CRD). The structure shows that RANKL trimer uses three equivalent grooves between two neighboring monomers to interact with three OPG-CRD monomers symmetrically. A loop from the CRD3 domain of OPG-CRD inserts into the shallow groove of RANKL, providing the major binding determinant that is further confirmed by affinity measurement and osteoclast differentiation assay. These results, together with a previously reported mouse RANKL/RANK complex structure, reveal that OPG exerts its decoy receptor function by directly blocking the accessibilities of important interacting residues of RANKL for RANK recognition. Structural comparison with TRAIL/death receptor 5 complex also reveals structural basis for the cross-reactivity of OPG to TRAIL.     

A new member of tumor necrosis factor ligand family, ODF/OPGL/TRANCE/RANKL, regulates osteoclast differentiation and function

Osteoclasts, the multinucleated giant cells that resorb bone, develop from monocyte-macrophage lineage cells. Osteoblasts or bone marrow stromal cells have been suggested to be involved in osteoclastic bone resorption. The recent discovery of new members of the tumor necrosis factor (TNF) receptor-ligand family has elucidated the precise mechanism by which osteoblasts/stromal cells regulate osteoclast differentiation and function. Osteoblasts/stromal cells express a new member of the TNF-ligand family "osteoclast differentiation factor(ODF)/osteoprotegerin ligand (OPGL)/TNF-related activation-induced cytokine (TRANCE)/receptor activator of NF-kB ligand (RANKL)" as a membrane associated factor. Osteoclast precursors which possess RANK, a TNF receptor family member, recognize ODF/OPGL/TRANCE/RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into osteoclasts in the presence of macrophage colony-stimulating factor. Mature osteoclasts also express RANK, and their bone-resorbingactivity is also induced by ODF/OPGL/TRANCE/RANKL which osteoblasts/stromal cells possess. Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF)/TNF receptor-like molecule 1 (TR1) is a soluble decoy receptor for ODF/OPGL/TRANCE/RANKL. Activation of NF-kB and c-Jun N-terminal kinase through the RANK-mediated signaling system appears to be involved in differentiation and activation of osteoclasts.     

RANKL与骨重建

骨是一种动态更新的组织,它不断进行骨吸收（bone resorption）与骨形成（bone formation）的平衡,这个过程称之为骨重建（bone remodeling）．核因子κB受体活化因子配体（receptor activator of nuclear factor κB ligand,RANKL）是骨吸收和骨形成耦联的关键,具有诱导破骨细胞（osteoclast, OC）生成、活化,抑制破骨细胞凋亡的作用．RANKL最初发现于活化的T细胞,但骨重建过程中RANKL主要来源于骨细胞、成骨细胞和骨髓基质细胞．RANKL/核因子κB受体活化因子（receptor activator of nuclear factor κB,RANK）/骨保护素（osteoprotegerin, OPG）信号通路在成骨细胞调控破骨细胞生成的过程中起着重要的调节作用,是维持骨重建平衡的关键．本文就RANKL及其在骨中的分子作用机制作一综述.     

High level secretory expression of murine OCIL by CHO cells and action of OCIL on osteoclast differentiation

Receptor activator of nuclear factor-kB ligand (RANKL), a well-known membrane-bound molecule expressed on osteoblasts and bone marrow stromal cells, is believed to induce osteoclast differentiation and activation by binding to the receptor activator of nuclear factor-kB (RANK), which is expressed on the surface of osteoclast lineage cells. This induction is inhibited by osteoprotegerin (OPG) that is secreted by osteoblast lineage and acts as a decoy receptor of RANKL. Currently the essential role of the OPG/RANKL/RANK system in the process of osteoclast maturation, as well as activation, has been well established, and the majority of bone resorption regulators control osteoclast formation and activation through their effects on this system and especially on the relative expression levels of RANKL and OPG [1].     

Zinc supplements and bone health: The role of the RANKL-RANK axis as a therapeutic target

BackgroundTo this day, empirical data suggests that zinc has important roles in matrix synthesis, bone turnover, and mineralization and its beneficial effects on bone could be mediated through different mechanisms. The influence of zinc on bone turnover could be facilitated via regulating RANKL/RANK/OPG pathway in bone tissue. Therefore, the aim of the study was to conduct a review to investigate the possible effect of the zinc mediated bone remodeling via RANKL/RANK/OPG pathway.MethodsA comprehensive systematic search was performed in MEDLINE/PubMed, Cochrane Library, SCOPUS, and Google Scholar to explore the studies investigating the effect of zinc as a bone remodeling factor via RANKL/RANK/OPG pathway regulation. Subsequently, the details of the pathway and the impact of zinc supplements on RANKL/RANK/OPG pathway regulation were discussed.ResultsThe pathway could play an important role in bone remodeling and any imbalance between RANKL/RANK/OPG components could lead to extreme bone resorption. Although the outcomes of some studies are equivocal, it is evident that zinc possesses protective properties against bone loss by regulating the RANKL/RANK/OPG pathway. There are several experiments where zinc supplementation resulted in upregulation of OPG expression or decreases RANKL level. However, the results of some studies oppose this.ConclusionIt is likely that sufficient zinc intake will elicit positive effects on bone health by RANKL/RANK/OPG regulation. Although the outcomes of a few studies are equivocal, it seems that zinc can exert the protective properties against bone loss by suppressing osteoclastogenesis via downregulation of RANKL/RANK. Additionally, there are several experiments where zinc supplementation resulted in upregulation of OPG expression. However, the results of limited studies oppose this. Therefore, aside from the positive role zinc possesses in preserving bone mass, further effects of zinc in RANKL/RANK/OPG system requires further animal/human studies.     

Mechanical stimulation effects on functional end effectors in osteoblastic MG-63 cells

Receptor activator of Nf-kappaB ligand (RANKL) and osteoprotegerin (OPG) have been implicated in bone metabolism. Specifically, the balance of these factors in conjunction with receptor activator of Nf-kappaB (RANK) is believed to be key in determining the rate of osteoclastogenesis and the net outcome of bone formation/resorption. While it is well accepted that mechanical loading in vivo affects bone formation/resorption and that alterations in the responsiveness of bone cells to mechanical loading have been implicated in metabolic bone diseases, the effect of in vitro mechanical loading on osteoblastic production of OPG and RANKL has not been extensively studied. Thus, in the current study, we developed an in vitro model to load human osteoblasts and studied levels of OPG, RANKL, PGE(2) and macrophage colony stimulating factor (M-CSF). We hypothesized that stimulating osteoblastic cells would increase the release of soluble OPG relative to RANKL favoring a bone-forming (and resorption-inhibiting) event. To accomplish this, we developed a small-scale loading machine that imparts via bending, well-defined substrate deformation to bone cells cultured on artificial substrates. Following 2h of loading and a 1h incubation period, media was collected and levels of soluble OPG, RANKL, PGE(2) and M-CSF were quantified using ELISA and western blotting. We found that mechanical loading significantly increased soluble OPG levels relative to RANKL at this 3h time point. Levels of soluble and cellular RANKL detected were not significantly affected by mechanical stimulation. The relative shift in abundance of OPG over RANKL associated with applied mechanical stimulation suggests the soluble OPG:RANKL ratio may be important in load-induced coupling mechanisms of bone cells.     

OPG, RANK and RANK ligand expression in thyroid lesions

Receptor activator of NF-kappaB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG) play essential roles in bone metabolism. RANKL binds to RANK, which is expressed by osteoclasts whereas OPG acts as its decoy receptor blocking the RANK-RANKL interaction. OPG/RANK/RANKL are produced by variety of tissues including epithelial and mesenchymal cells. However, the role of RANKL/OPG in thyroid pathophysiology remains unclear. The aim of this study was to determine the expression pattern of RANK/RANKL/OPG in primary neoplastic thyroid lesions and in lymph node metastases. 27 specimens from total thyroidectomy were studied by immunohistochemistry: 9 papillary carcinomas (PC), 9 medullary carcinomas (MC), 9 macrovesicular adenomas (MA). Immunohistochemical evidence of RANKL was found in 30 % of MC, 22% of PC while RANKL has never been detected in PC. The expression of RANK is closely related to RANKL. OPG was restricted to the cytoplasm of epithelial in 1 MA and 1 MC. In contrast to pathological tissues, any expression of OPG/RANK/RANKL was detected in healthy thyroid tissue. This work reveals for the first time that OPG/RANK/RANKL are expressed in the pathological thyroid gland by follicular cells, by malignant parafollicular cells as well as in metastatic lymph node microenvironment. Thus OPG/RANK/RANKL molecular triad might play a role during pathogenesis of follicular and parafollicular tumors.     

Detection and characterization of RANK ligand and osteoprotegerin in the thyroid gland

Receptor activator of NF-kappaB (RANK) ligand (RANKL) and osteoprotegerin (OPG) play essential roles in bone metabolism and immune responses. RANKL activates RANK, which is expressed by osteoclasts and dendritic cells (DC), whereas OPG acts as its decoy receptor. The role of RANKL and OPG in thyroid physiology is unclear. Northern analysis revealed pronounced OPG mRNA levels in normal human thyroid. By contrast, RANKL mRNA levels were most abundant in lymph node and appendix, and low in the thyroid. In the human thyroid follicular cell line XTC and in primary human thyroid follicular cells, OPG mRNA levels and protein secretion were upregulated by interleukin (IL)-1beta (33-fold), tumor necrosis factor (TNF)-alpha (eightfold), and thyrotropin (TSH) (threefold). RANKL mRNA was stimulated in XTC by IL-1beta and TNF-alpha, but inhibited by TSH. Conditioned medium harvested from IL-1beta-treated XTC (containing high concentrations of OPG) inhibited RANKL-induced CD40 upregulation and cluster formation of DC. OPG mRNA levels were three times more abundant in surgical thyroid specimens of Graves' disease as compared to other thyroid diseases. Our data suggest that RANKL and OPG are produced in the thyroid gland by thyroid follicular cells, are regulated by cytokines and TSH, and are capable of modulating dendritic cell functions. Thus, these cytokines may represent important local immunoregulatory factors involved in the pathogenesis of autoimmune thyroid diseases.     

Bone remodeling, particle disease and individual susceptibility to periprosthetic osteolysis

Bone remodeling is a tightly coupled process consisting of repetitive cycles of bone resorption and formation. Both processes are governed by mechanical signals, which operate in conjunction with local and systemic factors in a discrete anatomic structure designated a basic multicellular unit (BMU). The microenvironment around total joint arthroplasty is a dynamic and complex milieu influenced by the chemical and physical stimuli associated with servicing the prosthesis. A key factor limiting the longevity of the prosthesis is polyethylene wear, which induces particle disease, and this may lead to increased and prolonged activity of BMUs resulting in periprosthetic osteolysis. Several pathways regulating BMU function have been reported in the past, including RANKL/RANK/OPG/TRAF6, TNF-alpha/TNFR/TRAF1, and IL-6/CD126/JAK/STAT. Moreover, the expression and functional activity of all these molecules can be affected by variations in their genes. These may explain the differences in severity of bone defects or prosthetic failure between patients with similar wear rates and the same prosthesis. Simultaneously, this data strongly support the theory of individual susceptibility to prosthetic failure.