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The OPG / RANK-Ligand System

Bone Homeostasis

The homeostasis of the skeleton is characterized by a strict balance of bone formation and bone resorption. These bone remodeling processes occur in tandem at site-specific locations due to the action of mainly two cell types. Mesenchyme- derived osteoblasts are the driving force for the formation of mineralized bone. Bone resorption (osteolysis) is caused by haematopoietic and myelomonocytic derived osteoclasts. These opposing cells are connected via paracrine cell signalling loops. A shifted balance in favor of multinucleated osteoclasts causes bone destruction as observed in pathological conditions such as autoimmune arthritis, periodontitis, postmenopausal osteoporosis, Paget’s disease, Crohn’s disease and bone tumors.

RANK Signalling in Osteoclasts

RANKL

The receptor activator of nuclear factor κB ligand (RANKL [1]; OPGL [2]; TRANCE [3]; ODF [4]; TNFSF11) has been identified as an essential cytokine for the formation and activation of osteoclasts. It is a type II membrane protein with close homology to the TNF superfamily ligand members TRAIL (TNSF10), FasL (CD95L; CD178; TNSF6) and TNF α (TNSF2). RANKL contains a C-terminal ligand-binding and a transmembrane domain, but exists as both in a membrane-bound and soluble form.

RANK

RANKL binds to receptor activator of NF-κB (RANK [1]; TRANCE-R; ODAR; TNFRSF11A), a cloned member of the TNF receptor superfamily sharing the highest homology with CD40 (40%). RANK is a type I membrane protein that associates at the cell surface and contains four extracellular cysteine-rich pseudorepeats.

OPG [Osteoprotegerin]

The effects of RANKL are physiologically counterbalanced by the decoy receptor osteoprotegerin (OPG [5]; OCIF [6]; TR1 [7]; FDCR-1 [8]; TNFRSF11B), a naturally occuring member of the TNF receptor superfamily. OPG contains 4 cysteinerich domains, two death domain homologs and a heparin binding site, but does not have a transmembrane domain. The OPG gene product is a 401 aa polypeptide, which is signal peptidase-cleaved to 380 aa (44kDa), N-linked glycosylated (55kDa), and secreted as a disulfide-linked 110kDa homodimer. Dimerization is unusual among TNFRSF proteins, which typically associate as trimers. In vivo administration of OPG has been shown to increase bone mineral density and bone volume associated with a decrease in the number of active osteoclasts [5]. OPG binds also to the TNF Superfamily ligand TRAIL.

Signalling Pathways

In bone, RANKL is produced primarily by osteoblasts and bone marrow stromal cells. Binding of RANKL and macrophage/monocyte-colony forming factor (M-CSF) to their receptors (RANK and c-fms) on osteoclast precursors lead to osteoclast formation, while the binding of RANKL to its receptor (RANK) on mature osteoclasts triggers their activation and survival. Six key signalling pathways are activated upon binding to RANK: Akt/PKB (protein kinase B), Jun N-terminal kinase (JNK), extracellular signal regulated kinase (ERK), p38, nuclear factor κB (NF-κB) and nuclear factor of activated T cell c1 (NF-ATc1). One member of the tumor necrosis factor receptor associated factor (TRAF) family, TRAF6, binds to one of three cytoplasmic domains of RANK upon RANKL association, thereby activating the three mitogen activated protein kinase (MAPK) pathways (JNK, ERK and p38) and the NF-κB pathway. TRAF6 forms a complex with the scaffolding protein Cbl and phosphoinositide 3-kinase (PI(3)K) to activate the Akt/PKB pathway. Another signalling complex of TRAF6, TGF-β-activated kinase 1 (TAK1) and adapter protein TAB2 leads to the activation of the NF-κB and JNK pathways. Activation of p38 occurs upon its binding to TAB1 recruiting it to TRAF6/TAK1. The involvement of TRAF6 and the role of the cytoplasmic domains of RANK in the activation of the ERK pathway have yet to be defined. Transcription factor NF-ATc1 is a main regulator in osteoclastogenesis. NF-ATc1 is activated by RANKL and induces the expression of its own gene but is only fully activated if ITAM-mediated signals are present.

Effectors on RANK Signalling (ITAM-mediated Signals and Interferons)

RANK signalling is effected by further factors. Immunoreceptor tyrosine based activation motif (ITAM) is part of cytoplasmic domains of several transmembrane adapter molecules which link to immunoglobulin-like receptors. Two examples of these ITAM containing adapters are DNAX-activating protein 12 (DAP12) and Fc receptor common γ subunit (FcRγ) which have been shown to activate γ together calcium signalling, leading to the full activation of NFATc1 in osteoclast precursor cells [9]. While full activation of ITAMs requires signals from both the immunoreceptors and RANK signalling, the phosphorylating tyrosine kinase(s) activated by the RANKL signalling remain unknown. RANKL induces interferon-β (IFN-β) but not interferon-α (IFN-α) expression in osteoclast precursor cells. IFN-β strongly inhibits the osteoclast differentiation by interfering with the RANKL-induced expression of c-Fos. Activated T cells maintain bone homeostasis by counterbalancing the action of RANKL through production of IFN-γ. This cytokine induces rapid degradation of TRAF6 via signal transducer and activator of transcription

1 (STAT1) by proteasome activation and poly-ubiquitination.

Other Tumor Necrosis Factors (TNF-α, TRAIL and FasL)

As a major cytokine of inflammatory responses TNF-α plays a role in many diseases, such as postmenopausal osteoporosis, rheumatoid arthritis and periodontitis. It suppresses the recruitment of osteoblasts from progenitor cells and inhibits the expression of matrix protein genes necessary for bone mineralization. It also increases the production of interleukin-6 (IL-6) and M-CSF by osteoblasts, thereby indirectly promoting differentiation of osteoclasts and enhancing bone resorption. TNF-α can also modulate osteoclast formation and function by enhancing the expression of RANKL by osteoblasts and stromal cells. TNF-α is suspected to induce vitamin D resistance. The active metabolite of vitamin D3, calcitriol (1,25(OH)2D3) contributes to skeletal health by stimulating intestinal calcium absorbtion and also by directly regulating bone cell gene transcription. Osteoclast life span may be a keystone to bone remodeling. Because FasL, TRAIL and their receptors Fas (TNSFR6) and TRAIL-R1 to -R4 (TNSFR10A to 10D) are well known as cellular regulators of apoptosis, they may also be of interest in bone research. However, the precise role of these modulators in bone remodeling need further investigation.

Total RANKL, Soluble (human) ELISA Kit (APO-54N-016/1-KIO1)

Summary of Features

	New sensitive and specific assay for the quantitative determination of total
	human RANKL, a key factor in bone remodeling.
	Direct measurement of total OPG-complexed and-uncomplexed (free) human soluble RANKL from serum and cell culture supernatant [10].
	High Sensitivity: detection limit of 1.5 pg /ml.

Principle of the Kit

A 12 x 8-well microtiter plate is coated with a polyclonal antibody to OPG (human). Following addition of samples and OPG (human) recombinant, an incubation step with a biotin-labeled monoclonal antibody to RANKL (human) is performed. After washing, streptavidin horseradish-peroxidase is added. In a final step, the substrate tetramethylbenzidine (TMB) is used and the enzyme-catalyzed color conversion is monitored by an ELISA photometer.

Why measuring total RANKL instead of free RANKL

	Free RANKL in human serum is present at low concentrations that are difficult to detect.
	Total RANKL corresponds to free RANKL + RANKL complexed to the decoy receptor Osteoprotegerin (OPG).
	The RANKL complexed to OPG represents the major form of RANKL in serum (>99%)
	Measuring total RANKL give values in the nanogram range, while measuring free RANKL gives values 1000 times lower in the picogram range.
	Comparative tests indicate that both free and total RANKL levels correlate to same extent.

Literature Overview:

[1] A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function: D. M. Anderson, et al.; Nature 390, 175 (1997).

[2] Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation: D. L. Lacey, et al.; Cell 93, 165 (1998).

[3] TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells: B. R. Wong, et al.; J. Biol. Chem. 272, 25190 (1997).

[4] Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL: H. Yasuda, et al.; PNAS 95, 3597 (1998).

[5] Osteoprotegerin: a novel secreted protein involved in the regulation of bone density: W. S. Simonet, et al.; Cell 89, 309 (1997).

[6] Isolation of a novel cytokine from human fibroblasts that specifically inhibits osteoclastogenesis: E. Tsuda, et al.; BBRC 234, 137 (1997).

[7] TR1, a new member of the tumor necrosis factor receptor superfamily, induces fibroblast proliferation and inhibits osteoclastogenesis and bone resorption: B. S. Kwon, et al.; Faseb J. 12, 845 (1998).

[8] OPG/FDCR-1, a TNF receptor family member, is expressed in lymphoid cells and is up-regulated by ligating CD40: T. J. Yun, et al.; J. Immunol. 161, 6113 (1998).

[9] Costimulatory signals mediated by the ITAM motif cooperate with RANKL for bone homeostasis: T. Koga, et al.; Nature 428, 758 (2004).

[10] Effects of oral contraceptives on circulating osteoprotegerin and soluble RANK ligand serum levels in healthy young women: L. C. Hofbauer, et al.; Clin. Endocrinol. 60, 214 (2004).

Selected Latest Review Articles:

Osteoclast signalling pathways: H. C. Blair, et al.; BBRC 328, 728 (2005)/Regulatory roles and molecular signaling of TNF family members in osteoclasts: X. Feng; Gene 350, 1 (2005)/RANKing intracellular signaling in osteoclasts: X. Feng; IUBMB Life 57, 389 (2005)/Bone lesions in multiple myeloma--the OPG/RANK-ligand system: S. E. Goranov & V. S. Goranova-Marinova; Folia Med (Plovdiv) 46, 5 (2004)/A unified model for the action of leptin on bone turnover: J. O. Gordeladze & J. E. Reseland; J. Cell Biochem. 88, 706 (2003)/Signal transduction by receptor activator of nuclear factor kappa B in osteoclasts: Z. H. Lee & H. H. Kim; BBRC 305, 211 (2003)/Tumor necrosis factor-alpha: molecular and cellular mechanisms in skeletal pathology: M. S. Nanes; Gene 321, 1 (2003)/Inflammatory bone destruction and osteoimmunology: H. Takayanagi; J. Periodontal Res. 40, 287 (2005)/Central control of bone remodeling: S. Takeda; BBRC 328, 697 (2005)/Genetic regulation of osteoclast development and function: S. L. Teitelbaum & F. P. Ross; Nat. Rev. Genet. 4, 638 (2003)/Biology of the TRANCE axis: M. C. Walsh & Y. Choi; Cytokine Growth Factor Rev. 14, 251 (2003)