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。     

Inflammatory T cells rapidly induce differentiation of human bone marrow stromal cells into mature osteoblasts

Activated T cells secrete multiple osteoclastogenic cytokines which play a major role in the bone destruction associated with rheumatoid arthritis. While the role of T cells in osteoclastogenesis has received much attention recently, the effect of T cells on osteoblast formation and activity is poorly defined. In this study, we investigated the hypothesis that in chronic inflammation activated T cells contribute to enhanced bone turnover by promoting osteoblastic differentiation. We show that T cells produce soluble factors that induce alkaline phosphatase activity in bone marrow stromal cells and elevated expression of mRNA for Runx2 and osteocalcin. This data indicate that T cell derived factors have the capacity to stimulate the differentiation of bone marrow stromal cells into the osteoblast phenotype. RANKL mRNA was undetectable under any conditions in highly purified bone marrow stromal cells. In contrast, RANKL was constitutively expressed in primary osteoblasts and only moderately up-regulated by activated T cell conditioned medium. Interestingly, both bone marrow stromal cells and osteoblasts expressed mRNA for RANK, which was strongly up-regulated in both cell types by activated T cell conditioned medium. Although, mRNA for the RANKL decoy receptor, osteoprotegerin, was also up-regulated by activated T cell conditioned medium, it's inhibitory effects may be mitigated by a simultaneous rise in the osteoprotegerin competitor TNF-related apoptosis-inducing ligand. Based on our data we propose that during chronic inflammation, T cells regulate bone loss by a dual mechanism involving both direct stimulation of osteoclastogenesis, by production of osteoclastogenic cytokines, and indirectly by induction of osteoblast differentiation and up-regulation of bone turnover via coupling.     

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.     

Direct inhibition of human RANK+ osteoclast precursors identifies a homeostatic function of IL-1beta

IL-1β is a key mediator of bone resorption in inflammatory settings, such as rheumatoid arthritis (RA). IL-1β promotes osteoclastogenesis by inducing RANKL expression on stromal cells and synergizing with RANKL to promote later stages of osteoclast differentiation. Because IL-1Rs share a cytosolic Toll-IL-1R domain and common intracellular signaling molecules with TLRs that can directly inhibit early steps of human osteoclast differentiation, we tested whether IL-1β also has suppressive properties on osteoclastogenesis in primary human peripheral blood monocytes and RA synovial macrophages. Early addition of IL-1β, prior to or together with RANKL, strongly inhibited human osteoclastogenesis as assessed by generation of TRAP(+) multinucleated cells. IL-1β acted directly on human osteoclast precursors (OCPs) to strongly suppress expression of RANK, of the costimulatory triggering receptor expressed on myeloid cells 2 receptor, and of the B cell linker adaptor important for transmitting RANK-induced signals. Thus, IL-1β rendered early-stage human OCPs refractory to RANK stimulation. Similar inhibitory effects of IL-1β were observed using RA synovial macrophages. One mechanism of RANK inhibition was IL-1β-induced proteolytic shedding of the M-CSF receptor c-Fms that is required for RANK expression. These results identify a homeostatic function of IL-1β in suppressing early OCPs that contrasts with its well-established role in promoting later stages of osteoclast differentiation. Thus, the rate of IL-1-driven bone destruction in inflammatory diseases, such as RA, can be restrained by its direct inhibitory effects on early OCPs to limit the extent of inflammatory osteolysis.     

IL-17 promotes bone erosion in murine collagen-induced arthritis through loss of the receptor activator of NF-kappa B ligand/osteoprotegerin balance

IL-17 is a T cell-derived proinflammatory cytokine in experimental arthritis and is a stimulator of osteoclastogenesis in vitro. In this study, we report the effects of IL-17 overexpression (AdIL-17) in the knee joint of type II collagen-immunized mice on bone erosion and synovial receptor activator of NF-kappa B ligand (RANKL)/receptor activator of NF-kappa B/osteoprotegerin (OPG) expression. Local IL-17 promoted osteoclastic bone destruction, which was accompanied with marked tartrate-resistant acid phosphatase activity at sites of bone erosion in cortical, subchondral, and trabecular bone. Accelerated expression of RANKL and its receptor, receptor activator of NF-kappa B, was found in the synovial infiltrate and at sites of focal bone erosion, using specific immunohistochemistry. Interestingly, AdIL-17 not only enhanced RANKL expression but also strongly up-regulated the RANKL/OPG ratio in the synovium. Comparison of arthritic mice from the AdIL-17 collagen-induced arthritis group with full-blown collagen-arthritic mice having similar clinical scores for joint inflammation revealed lower RANKL/OPG ratio and tartrate-resistant acid phosphatase activity in the latter group. Interestingly, systemic OPG treatment prevented joint damage induced by local AdIL-17 gene transfer in type II collagen-immunized mice. These findings suggest T cell IL-17 to be an important inducer of RANKL expression leading to loss of the RANKL/OPG balance, stimulating osteoclastogenesis and bone erosion in arthritis.     

Protein expression and functional difference of membrane-bound and soluble receptor activator of NF-kappaB ligand: modulation of the expression by osteotropic factors and cytokines

A variety of humoral factors modulate the osteoclastogenesis. Receptor activator of NF-kappaB ligand (RANKL) expressed on osteoblast/stromal lineage cells plays a pivotal role to transduce an essential differentiation signal to osteoclast lineage cells through binding to its receptor, RANK, expressed on the latter cell population; however, the difficulty to detect RANKL protein expression hampers us in investigating the regulation of RANKL expression by humoral factors. To determine protein expression of RANKL, we have established a new method, named as a ligand-receptor precipitation (LRP) Western blot analysis, which can specifically concentrate the target protein by the use of specific binding characteristic between RANKL and RANK/osteoprotegrin (OPG). RANKL protein expression in the postnuclear supernatant was not detected by common Western blotting, but LRP Western blot analysis clearly showed that RANKL is produced as a membrane-bound protein on murine osteoblasts/stromal cells, and cleaved into a soluble form by metalloprotease. Cytokines stimulating the osteoclastogenesis, such as IL-1beta, IL-6, IL-11, IL-17, and TNF-alpha, increased the expression of RANKL with decrease of OPG expression in osteoblasts/stromal cells. In contrast, cytokines inhibiting the osteoclastogenesis, such as IL-13, INF-gamma, and TGF-beta1 suppressed the expression of RANKL and/or augmented OPG expression. Functional difference between membrane-bound and soluble RANKL was demonstrated, which showed that membrane-bound RANKL works more efficiently than soluble RANKL in the osteoclastogenesis developed from murine bone marrow cell culture. The present study indicates the usefulness of LRP Western blot analysis, which shows that the modulation of osteoclastogenesis by humoral factors is achieved, in part, by regulation of the expression of RANKL and OPG in osteoblast/stromal lineage cells.     

RANK, RANKL and OPG in inflammatory arthritis and periprosthetic osteolysis

Elucidation of the receptor activator of nuclear factor kappa B (RANK), its ligand (RANKL) and osteoprotegerin (OPG) as the final effectors of bone resorption has transformed our understanding of metabolic bone diseases and revealed novel therapeutic targets. Activation of the RANK-RANKL signaling pathway is directly responsible for dramatic focal erosions that are observed in inflammatory arthritis and aseptic loosening of orthopaedic implants. While these conditions share many features common to all metabolic bone disorders (e.g., osteoclastic resorption), they exhibit several unique properties, which are highlighted in this review. Most important is the relative inability of bisphosphonate therapy to inhibit osteolysis in joint inflammation and periprosthetic joint loosening and the unexpected effectiveness of anti-cytokine therapy in both rheumatoid and psoriatic arthritis. Herein, we provide a review of the role of RANK, RANKL and OPG in erosive arthritis and periprosthetic osteolysis and discuss the potential of anti-RANKL therapy for these conditions.     

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].     

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.     

The molecular mechanism of osteoclastogenesis in rheumatoid arthritis

Bone-resorbing osteoclasts are formed from hemopoietic cells of the monocyte–macrophage lineage under the control of bone-forming osteoblasts. We have cloned an osteoblast-derived factor essential for osteoclastogenesis, the receptor activator of NF-κB ligand (RANKL). Synovial fibroblasts and activated T lymphocytes from patients with rheumatoid arthritis also express RANKL, which appears to trigger bone destruction in rheumatoid arthritis as well. Recent studies have shown that T lymphocytes produce cytokines other than RANKL such as IL-17, granulocyte–macrophage colony-stimulating factor and IFN-γ, which have powerful regulatory effects on osteoclastogenesis. The possible roles of RANKL and other cytokines produced by T lymphocytes in bone destruction are described.     

Osteopontin as a positive regulator in the osteoclastogenesis of arthritis

We examined the role of osteopontin (OPN) in the osteoclastogenesis of arthritis using collagen-induced arthritis (CIA). Cells from arthritic joints of wild-type (OPN +/+) mice spontaneously developed bone-resorbing osteoclast-like cells (OCLs). The cultured cells showed an enhanced expression of receptor activator of nuclear factor kappaB ligand (RANKL) and a decreased expression of osteoprotegerin (OPG). The addition of OPG reduced the number of OCLs, indicating that the osteoclastogenesis depends on the RANK/RANKL/OPG system. The cells also produced OPN abundantly and anti-OPN neutralizing antibodies suppressed the development of OCLs. Moreover, the addition of OPN increased the expression of RANKL and augmented differentiation of OCLs from OPN-deficient (OPN -/-) cells. OPN, like the combination of 1alpha,25-dihydroxyvitamin D(3) and dexamethasone, also enhanced the RANKL expression and decreased OPG expression in a stromal cell line, ST2. These results suggest that OPN acts as a positive regulator in the osteoclastogenesis of arthritis through the RANK/RANKL/OPG system.     

Relevance of an in vitro osteoclastogenesis system to study receptor activator of NF-kB ligand and osteoprotegerin biological activities

Receptor activator of NF-kB Ligand (RANKL) is an essential requirement for osteoclastogenesis and its activity is neutralized by binding to the soluble decoy receptor osteoprotegerin (OPG). The purpose of this work was to study the effects of RANKL and OPG during osteoclastogenesis using the murine monocytic cell line RAW 264.7 that can differentiate into osteoclasts in vitro. RAW 264.7 cells plated at 10(4) cells/cm(2) and cultured for 4 days in the presence of RANKL represent the optimal culture conditions for osteoclast differentiation, with an up-regulation of all parameters related to bone resorption: tartrate resistant acid phosphatase (TRAP), calcitonin receptor (CTR), RANK, cathepsin K, matrix metalloproteinase (MMP)-9 mRNA expressions. RANKL and OPG biological effects vary according to the differentiation state of the cells: in undifferentiated RAW 264.7 cells, TRAP expression was decreased by OPG and RANKL, RANK expression was inhibited by OPG, while MMP-9 and cathepsin K mRNA expressions were not modulated. In differentiated RAW 264.7 cells, RANKL and OPG both exert an overall inhibitory effect on the expression of all the parameters studied. In these experimental conditions, OPG-induced MMP-9 inhibition was abrogated in the presence of a blocking anti-RANKL antibody, suggesting that part of OPG effects are RANKL-dependent.     

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.     

Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhances osteoclastogenesis

The signaling molecule Wnt regulates bone homeostasis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathways. Impairment of canonical Wnt signaling causes bone loss in arthritis and osteoporosis; however, it is unclear how noncanonical Wnt signaling regulates bone resorption. Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor (Ror) proteins. We showed that Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhanced osteoclastogenesis. Osteoblast-lineage cells expressed Wnt5a, whereas osteoclast precursors expressed Ror2. Mice deficient in either Wnt5a or Ror2, and those with either osteoclast precursor-specific Ror2 deficiency or osteoblast-lineage cell-specific Wnt5a deficiency showed impaired osteoclastogenesis. Wnt5a-Ror2 signals enhanced receptor activator of nuclear factor-κB (RANK) expression in osteoclast precursors by activating JNK and recruiting c-Jun on the promoter of the gene encoding RANK, thereby enhancing RANK ligand (RANKL)-induced osteoclastogenesis. A soluble form of Ror2 acted as a decoy receptor of Wnt5a and abrogated bone destruction in mouse arthritis models. Our results suggest that the Wnt5a-Ror2 pathway is crucial for osteoclastogenesis in physiological and pathological environments and represents a therapeutic target for bone diseases, including arthritis.     

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.     

IL-23 induces human osteoclastogenesis via IL-17 <Emphasis Type="Italic">in vitro</Emphasis>, and anti-IL-23 antibody attenuates collagen-induced arthritis in rats

This study demonstrates that IL-23 stimulates the differentiation of human osteoclasts from peripheral blood mononuclear cells (PBMC). Furthermore, in vivo blockade of endogenous IL-23 activity by treatment with anti-IL-23 antibody attenuates collagen-induced arthritis in rats by preventing both inflammation and bone destruction. IL-23 induced human osteoclastogenesis in cultures of PBMC in the absence of osteoblasts or exogenous soluble-receptor activator of NF-kappaB ligand (RANKL). This IL-23-induced osteoclastogenesis was inhibited by osteoprotegerin, anti-IL-17 antibody, and etanercept, suggesting that RANKL, IL-17, and TNF-alpha are involved. In addition, we found the ratio of production levels of IL-17 to those of IFN-gamma from activated human T cells was elevated at 1 to 10 ng/ml IL-23. The inductive effect of IL-17 and the inhibitory effect of IFN-gamma on osteoclastogenesis indicate that the balance of these two cytokines is particularly important. We also demonstrated that IL-23 administered at a later stage significantly reduced paw volume in rats with collagen-induced arthritis, in a dose-dependent manner. Furthermore, anti-IL-23 antibody reduced synovial tissue inflammation and bone destruction in these rats. These findings suggest that IL-23 is important in human osteoclastogenesis and that neutralizing IL-23 after onset of collagen-induced arthritis has therapeutic potential. Thus, controlling IL-23 production and function could be a strategy for preventing inflammation and bone destruction in patients with rheumatoid arthritis.     

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.     

重组可溶性核因子κB受体活化因子体外抑制甲状旁腺激素诱导的破骨细胞生成

新近发现的核因子κB受体活化因子配基（receptor activator of nuclear factor-κB ligand,RANKL）,核因子κB受体活化因子（receptor activator ofnuclear factor-κB,RANK）／护骨素（osteoprotegerin,OPG）细胞因子系统提高了对破骨细胞生物学和骨稳态分子水平的认识。RANKL与RANK之间的相互作用决定了破骨细胞的分化。本研究通过体外实验评价可溶性RANK （soluble RANK,sRANK）是否可作为RANKL的拈抗剂下调破骨细胞生成和骨吸收陷窝的形成。构建sRANK的原核表达载体,转化入大肠杆菌表达菌株Origami B（DE3）,成功表达了重组蛋白,亲和层析进行纯化。重组sRANK以剂量依赖方式抑制由甲状旁腺激素（parathyroid hormone,PTH）诱导的破骨细胞生成和骨吸收陷窝形成。RT-PCR实验证实,sRANK可显著抑制PTH刺激的小鼠骨髓细胞碳酸苷酶Ⅱ和抗酒石酸酸性磷酸酶mRNA的表达。结果表明,sRANK具有抗骨吸收功能,可能成为一种治疗以骨吸收加强为特征的骨疾病的新方法。     

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].