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.     

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.     

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.     

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。     

Physiology and pathophysiology of the RANKL/RANK system

The TNF family molecule RANKL and its receptor RANK are key regulators of bone remodeling, lymph node formation, and mammary gland development during pregnancy. RANKL and RANK are also expressed in the central nervous systems (CNS). However, the functional relevance of RANKL/RANK in the brain was entirely unknown. Recently, our group reported that the RANKL/RANK signaling pathway has an essential role in the central regulation of body temperature via the prostaglandin axis. This review discusses novel aspects of the RANKL/RANK system as key regulators of fever and female basal body temperature in the CNS.     

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.     

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.     

Expression pattern of receptor activator of NFκB (RANK) in a series of primary solid tumors and related bone metastases

Receptor activator of NFκB ligand (RANKL), RANK, and osteoprotegerin (OPG) represent the key regulators of bone metabolism both in normal and pathological conditions, including bone metastases. To our knowledge, no previous studies investigated and compared RANK expression in primary tumors and in bone metastases from the same patient. We retrospectively examined RANK expression by immunohistochemistry in 74 bone metastases tissues from solid tumors, mostly breast, colorectal, renal, lung, and prostate cancer. For 40 cases, tissue from the corresponding primary tumor was also analyzed. Sixty‐six (89%) of the 74 bone metastases were RANK‐positive and, among these, 40 (59.5%) showed more than 50% of positive tumor cells. The median percentage of RANK‐positive cells was 60% in primary tumors and metastases, without any statistically significant difference between the two groups (P = 0.194). The same percentage was obtained by considering only cases with availability of samples both from primary and metastasis. Our study shows that RANK is expressed by solid tumors, with high concordance between bone metastasis and corresponding primary tumor. These data highlight the central role of RANK/RANKL/OPG pathway as potential therapeutic target not only in bone metastasis management, but also in the adjuvant setting. J. Cell. Physiol. 226: 780–784, 2011. © 2010 Wiley‐Liss, Inc.     

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].     

Changes in RANKL/OPG/RANK gene expression in peripheral mononuclear cells following treatment with estrogen or raloxifene

The RANKL/OPG/RANK pathway is the key mediator of osteoclastogenesis. Mononuclear cells may be implicated in post-menopausal osteoporosis. The effect of estrogen or raloxifene on bone resorption and the expression of RANKL/OPG/RANK in peripheral blood mononuclear cells (PBMCs) was examined. Twenty-nine women with post-menopausal osteoporosis were treated with estrogen (HRT) or raloxifene for 12 months. Bone mineral density (BMD) was measured at baseline and at 12 months at the spine and hip. Serum C-terminal telopeptide (CTX) and OPG were measured at baseline and at 1, 3, 6 and 12 months. PBMCs were isolated from 17 women and changes in RANKL, OPG and RANK mRNA were determined. The effects of estrogen or raloxifene in PBMCs in vitro were also assessed. BMD increased following treatment (lumbar spine % change mean [S.E.M.]: 4.3% [0.9], p<0.001). Serum CTX decreased (6 months: -43.7% [6.0], p<0.0001). Serum OPG declined gradually (12 months: -26.4% [4.4], p<0.001). RANKL, OPG and RANK gene expression decreased (6 months: RANKL 50.0% [24.8] p<0.001, OPG: 21.7% [28] p<0.001, RANK: 76.6% [10.2] p=0.015). Changes in OPG mRNA correlated with changes in BMD (r=-0.53, p=0.027) and CTX (r=0.7, p=0.0044). Down-regulation in RANKL, OPG, RANK mRNA and reduction in bone resorption was also seen in vitro. These results suggest that the expression of RANKL/OPG/RANK in PBMCs are responsive to the slowing in bone turnover/remodeling associated with treatment with estrogen or raloxifene. Further confirmatory studies are needed.     

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.     

The OPG/RANKL/RANK system in metabolic bone diseases

The OPG/RANKL/RANK cytokine system is essential for osteoclast biology. Various studies suggest that human metabolic bone diseases are related to alterations of this system. Here we summarize OPG/RANKL/RANK abnormalities in different forms of osteoporoses and hyperparathyroidism. Skeletal estrogen agonists (including 17beta-estradiol, raloxifene, and genistein) induce osteoblastic OPG production through estrogen receptor-alpha activation in vitro, while immune cells appear to over-express RANKL in estrogen deficiency in vivo. Of note, OPG administration can prevent bone loss associated with estrogen deficiency as observed in both animal models and a small clinical study. Glucocorticoids and immunosuppressants concurrently up-regulate RANKL and suppress OPG in osteoblastic cells in vitro, and glucocorticoids are among the most powerful drugs to suppress OPG serum levels in vivo. As for mechanisms of immobilization-induced bone loss, it appears that mechanical strain inhibits RANKL production through the ERK 1/2 MAP kinase pathway and up-regulates OPG production in vitro. Hence, lack of mechanical strainduring immobilization may favor an enhanced RANKL-to-OPG ratio leading to increased bone loss. As for hyperparathyroidism, chronic PTH exposure concurrently enhances RANKL production and suppresses OPG secretion through activation of osteoblastic protein kinase A in vitro which would favour increased osteoclastic activity. In sum, the capacity for OPG to antagonize the increases in bone loss seen in many rodent models of metabolic bone disease implicates RANKL/OPG imbalances as the likely etiology and supports the potential role for a RANKL antagonist as a therapeutic intervention in these settings.     

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。     

TNF receptor type 1 regulates RANK ligand expression by stromal cells and modulates osteoclastogenesis

TNFalpha is a major osteoclastogenic cytokine and a primary mediator of inflammatory osteoclastogenesis. We have previously shown that this cytokine directly targets osteoclasts and their precursors and that deletion of its type-1 receptor (TNFr1) lessens osteoclastogenesis and impacts RANK signaling molecules. Osteoclastogenesis is primarily a RANK/RANKL-dependent event and occurs in an environment governed by both hematopoietic and mesenchymal compartments. Thus, we reasoned that TNF/TNFr1 may regulate RANKL and possibly RANK expression by stromal cells and osteoclast precursors (OCPs), respectively. RT-PCR experiments reveal that levels of RANKL mRNA in WT stromal cells are increased following treatment with 1,25-VD3 compared to low levels in TNFr1-null cells. Expression levels of OPG, the RANKL decoy protein, were largely unchanged, thus supporting a RANKL/OPG positive ratio favoring WT cells. RANK protein expression by OCPs was lower in TNFr1-null cells despite only subtle differences in mRNA expression in both cell types. Mix and match experiments of different cell populations from the two mice phenotypes show that WT stromal cells significantly, but not entirely, restore osteoclastogenesis by TNFr1-null OCPs. Similar results were obtained when the latter cells were cultured in the presence of exogenous RANKL. Altogether, these findings indicate that in the absence of TNFr1 both cell compartments are impaired. This was further confirmed by gain of function experiments using TNFr1- null cultures of both cell types at which exogenous TNFr1 cDNA was virally expressed. Thus, restoration of TNFr1 expression in OCPs and stromal cells was sufficient to reinstate osteoclastogenesis and provides direct evidence that TNFr1 integrity is required for optimal RANK-mediated osteoclastogenesis.     

Long chain polyunsaturated fatty acids alter membrane-bound RANK-L expression and osteoprotegerin secretion by MC3T3-E1 osteoblast-like cells

Inflammation triggers an increase in osteoclast (bone resorbing cell) number and activity. Osteoclastogenesis is largely controlled by a triad of proteins consisting of a receptor (RANK), a ligand (RANK-L) and a decoy receptor (osteoprotegerin, OPG). Whilst RANK is expressed by osteoclasts, RANK-L and OPG are expressed by osteoblasts. The long chain polyunsaturated fatty acid (LCPUFA) arachidonic acid (AA, 20:4n-6) and its metabolite prostaglandin E2 (PGE2), are pro-inflammatory and PGE2 is a potent stimulator of RANKL expression. Various LCPUFAs such as eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3) and gamma-linolenic acid (GLA, 18:3n-6) have anti-inflammatory activity. We aimed to determine if AA itself can stimulate RANKL expression and whether EPA, DHA and GLA inhibit RANKL expression in osteoblasts. MC3T3-E1/4 osteoblast-like cells were cultured under standard conditions with each of the LCPUFAs (5microg/ml) for 48h. Membrane-bound RANKL expression was measured by flow cytometry and OPG secretion measured by ELISA. In a second experiment, RANKL expression in MC3T3-E1/4 cells was stimulated by PGE2 treatment and the effect of EPA, DHA and GLA on membrane-bound RANKL expression and OPG secretion determined. The percentage of RANKL-positive cells was higher (p<0.05) than controls following treatment with AA or GLA but not after co-treatment with the cyclooxygenase inhibitor, indomethacin. DHA and EPA had no effect on membrane-bound RANKL expression under standard cell culture conditions. Secretion of OPG was lower (p<0.05) in AA-treated cells but not significantly different from controls in GLA, EPA or DHA treated cells. Treatment with prostaglandin E2 (PGE2) resulted in an increase (p<0.05) in the percentage of RANK-L positive cells and a decrease (p<0.05) in mean OPG secretion. The percentage of RANKL positive cells was significantly lower following co-treatment with PGE2 and either DHA or EPA compared to treatment with PGE2 alone. Mean OPG secretion remained lower than controls in cells treated with PGE2 regardless of co-treatment with EPA or DHA. Results from this study suggest COX products of GLA and AA induce membrane-bound RANKL expression in MC3T3-E1/4 cells. EPA and DHA have no effect on membrane-bound RANKL expression in cells cultured under standard conditions however both EPA and DHA inhibit the PGE2-induced increase in RANKL expression in MC3T3-E1/4 cells.     

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.     

Role of the OPG/RANK/RANKL triad in calcifications of the atheromatous plaques: comparison between carotid and femoral beds

Recent works demonstrated the difference of calcification genesis between carotid and femoral plaques, femoral plaques being more calcified. It has been clearly demonstrated that the molecular triad osteoprotegerin (OPG)/Receptor Activator of NFkB (RANK)/RANK Ligand (RANKL) exerts its activities in the osteoimmunology and vascular system. The aim of this study was to determine their expression and their potential role in calcifications of the atheromatous plaques located in two different peripheral arterial beds, carotid and femoral. The expression of OPG, RANK and RANKL was analyzed by immunochemistry in 40 carotid and femoral samples. Blood OPG and RANKL were quantified using specific ELISA assays. OPG staining was more frequently observed in carotid than in femoral plaques, especially in lipid core. Its expression correlated with macrophage infiltration more abundantly observed in carotid specimens. Surprisingly, serum OPG concentration was significantly lower in carotid population compared to femoral population while RANK and RANKL were equally expressed in both arterial beds. Carotid plaques that are less rich in calcium than femoral specimens, express more frequently OPG, this expression being correlated with the abundance of macrophages in the lesions. These data strengthen the key role played by OPG in the differential calcification in carotid and femoral plaques.     

Role of OPG/RANKL/RANK axis on the vasculature

Vascular calcification, a degenerative process considered in the past to be a passive procedure, has now been suggested to be related to ossification. Many proteins responsible for bone formation have been identified on the arterial wall. The OPG/RANKL/RANK axis, responsible for ossification and bone mineralization, seems to play a major role in vasculature and atherosclerosis. Mice lacking OPG gene present osteoporosis and arterial calcification, while overexpression of OPG gene leads to osteopetrosis. In the present review the latest knowledge related to the effects of the OPG/RANKL/RANK axis on vasculature, including atherosclerosis, will be analyzed. The clinical significance of circulating OPG and RANKL levels in vascular diseases will also be referred.