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

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Abstract

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-α/IL-1: interrelations in bone resorption pathophysiology. Cytokine Growth Factor Rev 2004;15:49–60].

Introduction

Bone is a specialized connective tissue formed by a mineralized matrix that confers it with elasticity and strength. Bone remodeling allows for adaptation to mechanical constraints and maintains homeostasis of phosphorus and calcium through coordinated phases of formation and resorption. Thus, bone remodeling involves synthesis of organic matrix by osteoblasts and bone resorption by osteoclasts. Osteoblasts differentiate from mesenchymal stem cells through a series of progenitor stages to form mature matrix-secreting osteoblasts and are progressively transformed into osteocytes [2]. Osteoblasts and osteocytes connected by gap junctions then constitute a cellular network, both on the bone surface and within the bone matrix. Osteoclasts are the main protagonists of bone resorption. If they are hematopoietic in origin and are closely related to macrophages, they also possess several characteristic features (multinucleation, highly polarized morphology, and numerous mitochondria). Osteoclasts resorb bone by attaching to the surface and then secreting protons into an extracellular compartment formed under their ruffled border. This secretion is necessary for bone mineral solubilization and the digestion of the organic matrix by acid proteases [3]. The other cell protagonists present in the bone microenvironnement (monocytes/macrophages, lymphocytes, and endothelial cells) also contribute to bone remodeling by direct contact with bone cells or by the release of soluble effectors. Thus, the numerous cellular protagonists involved in bone homeostasis are responsible for the connections that are established between bone tissue, the immune system, and the vascular compartment and allow for a better understanding of the associated pathology.

In 1965, Epker and Frost [4] demonstrated that the interactions between osteoblasts and osteoclasts are essential in bone remodeling. This equilibrium between osteoblast and osteoclast activities is tightly regulated by physical parameters (that is, mechanical stimulation) and numerous polypeptides (hormones, cytokines) [5]. Any disturbance between these effectors leads to the development of skeletal abnormalities, characterized by decreased (osteoporosis) or increased (osteopetrosis) bone mass. Increased osteoclast activity is observed in many osteopathic disorders, including post-menopausal osteoporosis, Paget's disease, primary bone tumors, lytic bone metastases, multiple myeloma, and rheumatoid arthritis, leading to increased bone resorption and a loss of bone mass. Osteoblasts control bone-resorbing activities of osteoclasts and are also clearly involved in osteoclast differentiation. Thus, coculture of osteoblast (bone marrow stromal cells) and osteoclast precursors (spleen or bone marrow cells) results in the formation of functional osteoclasts. In contrast, when both cell populations are separated by a selective membrane permeable to soluble effectors but not to cells, no osteoclasts are formed [6], [7]. These studies demonstrated for the first time that cell–cell contacts are required for the induction of osteoclastogenesis by osteoblastic cells. Moreover, these data imply that osteoblasts express ligands on their membrane that are recognized by receptors on the surface of osteoclasts. All of these observations drove many scientists to look for the effectors responsible for osteoblast-osteoclast interactions. Thus, in 1997 and 1998, research led to the identification of a novel set of cytokines within the TNF family that are required for the control of bone remodeling [8], [9], [10], [11].

The present manuscript is an extension of the earlier review recently published by Kwan Tat et al. [1] and focuses on the involvement of osteoprotegerin (OPG), of the receptor activator of nuclear factor κB ligand (RANKL), and of its cognate receptor RANK in the orchestration of pathophysiological bone remodeling (structure, production, regulation, interactions, biological activities) and their potential implications in benign and malignant human pathology.

Section snippets

The discovery of the molecular triad OPG/RANK/RANKL was a major event of the past decade in bone biology

Working on the identification of TNF receptor-related molecules with potential therapeutic interests, the Amgen group demonstrated the existence of a truncated TNF receptor-like molecule responsible for the marked osteopetrosis phenotype when overexpressed in transgenic mice [8]. Complementary experiments revealed that this osteopetrosis is associated with a decrease in osteoclastogenesis and in osteoclast activation. This bone-protecting molecule was named osteoprotegerin (OPG).

The wide range of cells which produce OPG, RANK and RANKL allows definition of three main biological systems controlled by this triad

While OPG, RANK and RANKL are produced by numerous cell types and a variety of tissues (Table 2), their expression pattern targets three main biological systems where the molecular triad could be more specifically involved: the osteoarticular, immune, and vascular systems (Fig. 2). Transgenic and knockout mice clearly revealed the potential involvement of these effectors in the three biological systems. Thus, mice deficient in OPG exhibit a decrease of total bone density and a high incidence of

OPG/RANK/RANKL are key molecules for osteoclastic differentiation but can be shunted by other molecular mediators

OPG expression and RANKL expression are modulated by numerous osteotropic agents (Table 3). Among them, OPG expression is positively regulated by estrogen, TNF-α, GH and TGF-β, and negatively by PTH and glucocorticoids. RANKL is also strongly modulated by the same effectors controlling OPG. Contrasting with OPG and RANKL, the regulation of RANK expression is more restricted to immune cells. Indeed, if RANK expression is controlled on dendritic cells by CD40L and via TCR engagement on T

TRAF6 plays a key role in the control of osteoclastic biology by OPG/RANK/RANKL

Within the OPG/RANK/RANKL triad, soluble and membrane forms of RANKL expressed by osteoblasts exert their activities through binding to their RANK receptor on osteoclasts [18]. The binding of RANKL to the extracellular RANK domain leads to the expression of specific genes involved during osteoclast differentiation, bone resorption, and osteoclast survival (Fig. 3). The initial step in RANK signaling is the binding to the TNF receptor-associated factor (TRAF) adaptator proteins within the

Osteoclastic differentiation is controlled by complex interactions between OPG, RANK, RANKL, and other secondary modulators

OPG, RANK, and RANKL molecules are characterized by their capability to form homo- and hetero-multimerized complexes (Fig. 4). Thus, while the OPG monomer is biologically active, OPG dimer formation is required to elicit full biological activity in vitro and in vivo [8]. Similarly, RANKL can form a homotrimer, as revealed by the study of their crystal structures. In this context, Lam et al. [23] proposed a model in which trimeric RANKL binds to a trimeric RANK complex, in which the

The OPG/RANK/RANKL triad is implicated in many non-malignant pathological conditions

The crucial role of OPG/RANK/RANKL in bone homeostasis and in cardiovascular functions allowed the identification of genetic mutations in RANK or OPG genes, which activate RANK or alter the interactions between RANKL and OPG, leading to hyperphosphatasia or bone abnormalities and to various vascular morphologies (Table 4). Thus, osteolytic and non-osteolytic hyperphosphatasia have been associated with an activating mutation in exon 1 of the gene encoding RANK (18 bp duplication in position 84)

OPG/RANK/RANKL involvement in malignant pathology

The dysregulation of the functional equilibrium in the OPG/RANK/RANKL triad is responsible for the osteolysis associated with malignant tumors and for the development of such tumors in bone sites (Table 5). RANKL has already been detected in several tumor cells and can be considered as a key factor involved in bone remodeling associated with bone metastases [209], [217], [219], [230], [231]. Moreover, although several primary melanoma and breast cancer cells did not express RANKL transcripts

Therapeutic strategies based on the OPG/RANK/RANKL triad

The detection of human OPG in the sera of rats gavaged with human milk affirms the key role of OPG during bone growth and the relevance of its involvement for the natural prevention of immune and bone disorders [229]. In light of the data described above, novel strategic treatments for bone loss are emerging that are based on an understanding of the functional status of the OPG/RANK/RANKL triad. Three therapeutic strategies have been envisaged; these involve: (1) cytokines and their soluble

Acknowledgements

This work was supported by a CReS INSERM no. 4CR06F, by a grant from the French Ministry of Research and Technology (TS/0220044) and by a grant from the Loire-Atlantique Committee of the Ligue Contre le Cancer. S. Théoleyre and Y. Wittrant, respectively, received a fellowship from the Loire-Atlantique Committee of the Ligue Contre le Cancer and from the Région of Pays de Loire.

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