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Keywords:

  • osteoclastogenesis;
  • TNF;
  • cytokines;
  • osteoporosis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. OSTEOPROTEGERIN LIGAND/OSTEOCLAST DIFFERENTIATION FACTOR
  5. THE RECEPTOR FOR OPG-L/ODF
  6. OPG/OCIF
  7. PARACRINE REGULATION OF OSTEOCLASTOGENESIS: INTEGRATION OF MECHANISMS
  8. RELEVANCE TO METABOLIC BONE DISORDERS
  9. POTENTIAL THERAPEUTIC USES OF OPG
  10. Acknowledgements
  11. REFERENCES

Although multiple hormones and cytokines regulate various aspects of osteoclast formation, the final two effectors are osteoprotegerin ligand (OPG-L)/osteoclast differentiation factor (ODF), a recently cloned member of the tumor necrosis factor superfamily, and macrophage colony–stimulating factor. OPG-L/ODF is produced by osteoblast lineage cells and exerts its biological effects through binding to its receptor, osteoclast differentiation and activation receptor (ODAR)/receptor activator of NF-κB (RANK), on osteoclast lineage cells, in either a soluble or a membrane-bound form, the latter of which requires cell-to-cell contact. Binding results in rapid differentiation of osteoclast precursors in bone marrow to mature osteoclasts and, at higher concentrations, in increased functional activity and reduced apoptosis of mature osteoclasts. The biological activity of OPG-L/ODF is neutralized by binding to osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF), a member of the TNF-receptor superfamily that also is secreted by osteoblast lineage cells. The biological importance of this system is underscored by the induction in mice of severe osteoporosis by targeted ablation of OPG/OCIF and by the induction of osteopetrosis by targeted ablation of OPG-L/ODF or overexpression of OPG/OCIF. Thus, osteoclast formation may be determined principally by the relative ratio of OPG-L/ODF to OPG/OCIF in the bone marrow microenvironment, and alterations in this ratio may be a major cause of bone loss in many metabolic disorders, including estrogen deficiency and glucocorticoid excess. That changes in but two downstream cytokines mediate the effects of large numbers of upstream hormones and cytokines suggests a regulatory mechanism for osteoclastogenesis of great efficiency and elegance.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. OSTEOPROTEGERIN LIGAND/OSTEOCLAST DIFFERENTIATION FACTOR
  5. THE RECEPTOR FOR OPG-L/ODF
  6. OPG/OCIF
  7. PARACRINE REGULATION OF OSTEOCLASTOGENESIS: INTEGRATION OF MECHANISMS
  8. RELEVANCE TO METABOLIC BONE DISORDERS
  9. POTENTIAL THERAPEUTIC USES OF OPG
  10. Acknowledgements
  11. REFERENCES

Maintainance of skeletal integrity requires a dynamic balance between bone formation and bone resorption that is fine tuned by a complex network of calcitropic hormones and cytokines.(1–3) The pool size of active osteoclasts is determined by the net effects of differentiation and fusion of osteoclast precursors and of the activity and rate of apoptosis of active osteoclasts.(1–4) Various cytokines secreted by osteoblast lineage cells (interleukin [IL]-1, tumor necrosis factor [TNF], IL-6, IL-11, and transforming growth factor β [TGF-β]), 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3), and glucocorticoids have been shown to play a role in the differentiation of pluripotent osteoclast progenitors into mature multinucleated osteoclasts (osteoclastogenesis).(1–4) However, the requirement of “feeder cells” and cell-to-cell contact of these cells with preosteoclasts suggested that additional unidentified factors were also required.(1–7)

The recent discovery of osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF),(8,9) and the subsequent identification of its cognate ligand, OPG ligand (OPG-L)/osteoclast differentiation factor (ODF),(10,11) has illuminated the molecular mechanisms of osteoclast differentiation and activation, and has created a new paradigm for the biology of the osteoclast (for abbreviations, see glossary in Table 1). Recent studies suggest that OPG-L/ODF is the missing factor needed for the completion of osteoclastogenesis and that its activity is modulated by the ratio of OPG-L/ODF to OPG/OCIF. Here we review the structure, expression, and regulation of OPG-L/ODF and OPG/OCIF, their effects on bone and mineral metabolism, and their potential role in the pathogenesis and treatment of various metabolic bone diseases.

Table Table 1. Glossary of Abbreviations Used
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OSTEOPROTEGERIN LIGAND/OSTEOCLAST DIFFERENTIATION FACTOR

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. OSTEOPROTEGERIN LIGAND/OSTEOCLAST DIFFERENTIATION FACTOR
  5. THE RECEPTOR FOR OPG-L/ODF
  6. OPG/OCIF
  7. PARACRINE REGULATION OF OSTEOCLASTOGENESIS: INTEGRATION OF MECHANISMS
  8. RELEVANCE TO METABOLIC BONE DISORDERS
  9. POTENTIAL THERAPEUTIC USES OF OPG
  10. Acknowledgements
  11. REFERENCES

Identification and characterization

The OPG-L/ODF was identified independently by two groups.(10,11) OPG-L/ODF was found to be identical with two previously cloned members of the TNF ligand family—TNF-related activation-induced cytokine (TRANCE), an early gene that is induced by stimulation of the T-cell receptor(12) and receptor activator of nuclear factor (NF)-κB ligand (RANKL), a stimulatory factor for dendritic cells.(13)

The human OPG-L/ODF is a polypeptide of 317 amino acids that is closely related to the TNF-related apoptosis-inducing ligand (TRAIL; 20–34% homology), to the CD40 ligand (28% homology), and to the Fas ligand (19% homology).(10–13) Human OPG-L/ODF is a type II membrane protein that lacks a signal peptide and has a cytoplasmic domain at the N terminus (residues 1–48), a transmembrane domain (residues 49–69), and an extracellular region at the C terminus (residues 70–317) that contains the active ligand site (residues 158–317) with 10 β-sheet–forming sequences present in TNF ligand family members.(10–13) OPG-L/ODF exists in two biologically active forms, a cellular, membrane-bound form (40–45 kDa) and a soluble form (31 kDa) derived posttranslationally by cleavage at positon 140 or 145.(10) The human OPG-L/ODF gene has been localized to chromosome 13q14.(12,13)

The promoter of the murine OPG-L/ODF gene has recently been shown to contain a response element for Cbfa-1,(14) a transcription factor essential for commitment to the osteoblastic differentiation pathway. This suggests a molecular mechanism for the previously observed coupling of osteoblastogenesis and osteoclastogenesis. Consistent with this hypothesis, embryonic mice deficient in Cbfa-1 are also deficient in OPG-L/ODF messenger RNA (mRNA) expression in the developing long bones, and calvarial cells from these mice have an impaired ability to support osteoclastogenesis.(15)

Expression and regulation

The steady-state levels of OPG-L/ODF mRNA are highest in skeletal (trabecular bone, bone marrow) and in lymphoid tissues (lymph node, thymus, spleen, fetal liver, Peyer's patches) that are active in mediating the immune response. However, lower levels are found in heart, skeletal muscle, lung, stomach, placenta, and thyroid gland.(10–13) Consistent with this tissue distribution, OPG-L/ODF mRNA has been detected in various stromal cell lines (ST-2, MC3T3-E1, hMS, primary human marrow stromal cells), osteosarcoma cell lines (ROS, MG-63), and primary murine osteoblasts.(10–13,16,17) In addition, high OPG-L/ODF mRNA levels are also present in various primary and transformed murine lymphoid cell systems, and are correlated with the content of T lymphocytes.(12,13) In fetal murine bone, OPG-L/ODF is mainly expressed by primitive mesenchymal cells adjacent to the cartilaginous anlagen, by hypertrophic chondrocytes, and in regions of primary ossification and modeling.(10) In adult murine bone, OPG-L/ODF is most abundant at the growth plate and the metaphyseal periosteum.(10) In situ hybridization localized high OPG-L/ODF mRNA expression to the white splenic pulp, the medulla of the thymus, the cortical areas of lymph nodes, and intestinal lymphoid patches.(10)

OPG-L/ODF mRNA steady-state levels in osteoblastic lineage cells are up-regulated by various calcitropic hormones and cytokines including dexamethasone, 1α,25-(OH)2D3, IL-1β, IL-11, TNF-α, parathyroid hormone (PTH), and prostaglandin (PG) E2,(11,17) whereas transforming growth factor β (TGF-β) suppresses them(18) (Table 2).

Table Table 2. Regulation of OPG-L/ODF and OPG/OCIF by Calcitropic Hormones and Cytokines
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Biological effects

In bone, OPG-L/ODF stimulates osteoclast differentiation,(10,11,19,20) enhances the activity of mature osteoclasts,(10,21,22) and inhibits osteoclast apoptosis:(21) the net effect of these actions is to expand the pool of activated osteoclasts (Fig. 1). In the presence of low levels of the permissive factor, macrophage colony–stimulating factor (M-CSF; also termed colony-stimulating factor 1) OPG-L/ODF is both necessary and sufficient for the complete differentiation of pluripotent osteoclast precursor cells into mature osteoclasts, and obviates the need of other cytokines, cofactors, or coculture systems.(10,11,19,20) In fact, the presence of both a cellular and a soluble form of OPG-L/ODF(10) explains some of the previous observations regarding the requirements for osteoclastogenesis.(1–4) The requirement for “feeder cells” is explained by production of OPG-L/ODF. Direct cell-to-cell contact with stromal cells would allow continuous exposure of the membrane-bound OPG-L/ODF to osteoclast precursors, whereas the soluble form would explain the stimulatory effect of conditioned medium harvested from stromal cells and osteoblasts. Recently, a specific metalloprotease has been isolated that cleaves membrane-bound OPG-L/ODF and releases it in the soluble form,(23) raising the possibility that the ratio of the two forms may be regulated. Interestingly, M-CSF, the other obligatory requirement for osteoclastogenesis, also exists in membrane-bound and soluble forms.(24,25)

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Figure FIG. 1. Control of osteoclast functions by OPG-L/ODF and OPG/OCIF. M-CSF, macrophage colony–stimulating factor; OCIF, osteoclastogenesis inhibitory factor; ODF, osteoclast differentiation factor; OPG, osteoprotegerin; OPG-L, osteoprotegerin ligand. Note that all steps of osteoclast formation and function are regulated by the OPG-L/OPG ratio including initiation of differentiation, fusion of preosteoclasts to form active mature osteoclasts, regulation of osteoclast function, and osteoclast apoptosis. M-CSF is required only for the initiation of differentiation by uncommitted osteoclast precursors in bone marrow.

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It is important to note that animals injected with osteoclastogenic doses of OPG-L/ODF only form osteoclasts in bone and not in other tissues that would also likely have permissive levels of M-CSF.(10) This observation implies that there may be other factor(s) present in the bone microenvironment (matrix, adhesion molecules, etc.) that are necessary for the differentiation of osteoclasts from osteoclast precursor cells. Whatever these factors may be, they are either present or mimicked by other factors in the in vitro culture models that have been used to characterize the actions of OPG-L/ODF.

Several studies have clearly established that OPG-L/ODF increased various osteoclastic differentiation markers in vitro, including activity of tartrate-resistent acid phosphatase (TRAP), number and percentage of TRAP-positive cells, and the expression of β3-integrin, cathepsin K, c-src, and calcitonin and vitronectin receptors.(10,11,19,20) Promotion of osteoclastogenesis by OPG-L/ODF was dose dependent (half-maximal effect at 1 ng/ml), specifically antagonized by coadministration of the soluble receptor antagonist OPG/OCIF, and functionally relevant as assessed by the ability of the osteoclasts to generate resorption lacunae.(10,11,19,20) Stimulation of osteoclastogenesis by OPG-L/ODF has been shown in both murine marrow cells and in human peripheral blood monocytes (which are believed to contain a subset of circulating osteoclast precursors),(19,20) and complete differentiation required between 7 (murine) and 11 (human) days.(19)

In addition to stimulating osteoclastogenesis, OPG-L/ODF (at the higher concentration of 1–10 ng/ml) directly stimulates osteoclastic pit formation in bovine bone slices(10) and45Ca release by fetal murine osteoclasts in long bone cultures.(26) A recent study(22) has shown that OPG-L/ODF in the absence of M-CSF also stimulated mature, fully differentiated osteoclasts to rapidly undergo ultrastructural changes (rearrangements of their actin rings) and to induce multiple cycles of bone resorption. In addition, the number and density of single, isolated resorption areas generated by mature osteoclasts were also stimulated by OPG-L/ODF administration.(22)

Direct administration of recombinant OPG-L/ODF to normal mice resulted in a dose-dependent, severe hypercalcemia within 1 day.(10) Mice treated with OPG-L for 3 days had marked bone loss associated with large, more differentiated osteoclasts, a doubling of the resorption surface, but no change in osteoclast number, suggesting that these effects resulted from an increased activation of existing osteoclasts rather than increased osteoclast formation.(10) In contrast, cotreatment of these OPG-L/ODF-treated mice with OPG/OCIF(10) and of hypercalcemic rats [induced by thyroparathyroidectomy and susequent treatment with PTH or 1α,25-(OH)2D3] with OPG/OCIF(27) normalized serum calcium levels.

In addition to effects on bone and mineral metabolism, OPG-L/ODF (which was initially identified by immunologists as TRANCE(12) or RANKL(13)) has a variety of effects on T-cell and dendritic cell function. TRANCE stimulated the activation of c-Jun N-terminal kinase (JNK) in T cells(12) and was found to bind to the surface of mature dendritic cells.(28) Moreover, TRANCE inhibited apoptosis of dendritic cells by increasing the expression of the antiapoptotic molecule bcl-xL.(28) Of note, RANKL was found to induce cluster formation by dendritic cells and to promote the allostimulatory capacity of dendritic cells on T cells as well as cytokine-activated T cell growth.(13)

OPG-L/ODF knock-out mice have provided important insights into both the skeletal and immunological functions of OPG-L in vivo.(29) These mice have severe osteopetrosis with shortened club-shaped bones and impaired tooth eruption, and they lack mature osteoclasts.(29) As a result of an accumulation of cartilage and bone, the marrow cavities of long bones are narrowed, leading to compensatory extramedullary hematopoiesis.(29) Interestingly, mice deficient in cbfa-1, which interacts with the OPG-L gene promoter,(14,15) also have narrow bone marrow cavities and associated extramedullary hematopoiesis.(30) Hematopoietic progenitor cells derived from OPG-L/ODF-deficient mice can develop into osteoclasts when exposed to recombinant OPG-L/ODF; however, stromal cells from the OPG-L/ODF-deficient mice do not support osteoclast formation when wild-type progenitors are used. These two pieces of data show that OPG-L/ODF is critical for osteoclast differentiation and further indicate that the OPG-L/ODF mice lack osteoclasts because of an intrinsic stromal cell defect as opposed to a defect in hematopoietic osteoclast progenitors.(29) In addition, OPG-L/ODF knockout mice, but not transgenic mice overexpressing OPG/OCIF,(8) have defects in B- and T-lymphocyte maturation, thymic hypoplasia, and lymph node agenesis.(29)

THE RECEPTOR FOR OPG-L/ODF

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. OSTEOPROTEGERIN LIGAND/OSTEOCLAST DIFFERENTIATION FACTOR
  5. THE RECEPTOR FOR OPG-L/ODF
  6. OPG/OCIF
  7. PARACRINE REGULATION OF OSTEOCLASTOGENESIS: INTEGRATION OF MECHANISMS
  8. RELEVANCE TO METABOLIC BONE DISORDERS
  9. POTENTIAL THERAPEUTIC USES OF OPG
  10. Acknowledgements
  11. REFERENCES

Identification and characterization

The receptor for OPG-L/RANKL, receptor activator of NF-κB (RANK) is a type I transmembrane protein.(13) The human RANK (616 amino acids) has a signal peptide (28 amino acids), an N-terminal extracellular domain (184 amino acids), a short transmembrane domain (21 amino acids), and a large C-terminal cytoplasmic domain (383 amino acids).(13) The gene encoding RANK has been localized to human chromosome 18q22.1.(13) The extracellular domain of RANK contains four cysteine-rich pseudorepeats and two N-glycosylation sites,(13,31) a characteristic feature of members of the TNFR superfamily, including OPG. RANK has a 40% homology with CD40.(13) Although RANK was originally described as a receptor on T cells and dendritic cells,(13) Hsu et al.(32) have recently shown that it is identical to the receptor located on osteoclasts that binds OPG-L and renamed it osteoclast differentiation and activation receptor (ODAR).

Expression and regulation

Whereas mRNA expression of ODAR/RANK has been detected in a variety of tissues, including skeletal muscle, liver, small intestine, colon, thymus, and adrenal gland, as well as osteoclastic, lymphocytic, and nonlymphocytic cells, the distribution of ODAR/RANK is restricted to osteoclasts, dendritic cells, B- and T-cell lines, and fibroblasts.(13,32,33) Among bone cells, ODAR/RANK is found only in osteoclast lineage cells.(32) In dendritic cells, ODAR/RANK expression is stimulated by CD40 ligand, whereas ODAR/RANK expression by T cells is stimulated by IL-4 and TGF-β.(13) It is not yet clear how ODAR/RANK is regulated in osteoclastic cells.

Biological effects and signal transduction

OPG-L/ODF binds to ODAR/RANK with high specificity and affinity. This activates a cascade of intracellular events, including interaction with TNFR-associated factor (TRAF) family members, activation of the transcriptional factor NF-κB, and stimulation of the protein kinase JNK.(13,31,32,34) Stimulating antibodies targeting the extracellular domains of ODAR/RANK mimicked the action of OPG-L/ODF and promoted osteoclastogenesis,(32,33) whereas inhibitory Fab fragments of this polyclonal antibody or a soluble ODAR/RANK blocked osteoclastogenesis by competing with OPG-L/ODF for cell-bound ODAR/RANK.(33) In addition, transgenic mice overexpressing soluble ODAR/RANK had a phenotype of decreased osteoclastogenesis, decreased bone resorption, and osteopetrosis, similar to that of the OPG-L/ODF-deficient mice.(32) Of interest, double-knockout mice deficient in both NF-κB1 and NF-κB2 also had impaired osteoclastogenesis and osteopetrosis,(35) highlighting the importance of NF-κB as a key transcription factor in the signaling pathway of OPG-L/ODF. Furthermore, targeted ablation of c-fos, a transcription factor that heterodimerizes with activated c-jun to modulate gene activation, also leads to a defect in osteoclast differentiation,(36,37) supporting the concept that both the NF-κB and JNK pathways are important effectors in the osteoclast differentiation pathway.

Recent studies have shown that the intracellular adaptor protein family, TRAF is involved in NF-κB and JNK activation by the activated ODAR/RANK. This family consists of six cytoplamic zinc finger proteins (TRAF1–TRAF6) that regulate cell proliferation and apoptosis by activating NF-κB and other transcriptional factors.(38) The intracellular domain of human ODAR/RANK contains three potential TRAF-binding domains,(31) which were found to interact with TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6.(31,34,39) The TRAFs differentially regulate either NF-κB activation, JNK stimulation, or both.(28,31,34,39,40) Mutational analyses of ODAR/RANK have shown that the C-terminal 85 amino acids (530–616) of human ODAR/RANK bind TRAF2, TRAF5, and TRAF6 and are essential for NF-κB activation,(31) and that NF-κB activation (and also JNK stimulation) is induced when TRAF2, TRAF5, and TRAF6 are overexpressed.(40) By contrast, ODAR/RANK-induced NF-κB activation is suppressed in cells carrying dominant negative forms of TRAF2, TRAF5, TRAF6, and TRAF-interacting protein, the endogenous inhibitor of TRAF2.(34) Moreover, the TRAF6-binding site (340–421)(39) and an intact zinc finger of the TRAFs(40) are required for NF-κB activation. As noted by Galibert et al.,(39) there appear to be discrepancies regarding the location of some of the TRAF-binding sites and the relevance of upstream ODAR/RANK sequences in NF-κB activation among some investigators. The reasons for these differences are unclear and may reflect differences in the experimental systems used. Of note, targeted ablation of TRAF-6 in knockout mice resulted in osteopetrosis and impaired tooth eruption.(41) Although osteoclast differentiation appears to be normal in vivo, osteoclasts from TRAF-6–deficient mice do not form ruffled borders, indicating that TRAF-6 may be important for osteoclast activation.(41) In further support of this, a recent study has identified a novel TRAF-6 interaction motif of ODAR/RANK that is both necessary and sufficient for ODAR/RANK-induced NF-κB activation.(42) It is unclear whether differences in TRAF profiles or modulation of TRAFs may alter the responsiveness of osteoclastic cells to OPG-L/ODF. In addition, the calcitropic hormones and cytokines regulating the concentration and affinity ODAR/RANK for OPG-L/ODF have not been systematically studied as yet.

OPG/OCIF

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. OSTEOPROTEGERIN LIGAND/OSTEOCLAST DIFFERENTIATION FACTOR
  5. THE RECEPTOR FOR OPG-L/ODF
  6. OPG/OCIF
  7. PARACRINE REGULATION OF OSTEOCLASTOGENESIS: INTEGRATION OF MECHANISMS
  8. RELEVANCE TO METABOLIC BONE DISORDERS
  9. POTENTIAL THERAPEUTIC USES OF OPG
  10. Acknowledgements
  11. REFERENCES

Identification and characterization

OPG/OCIF was identified independently by two groups(8,9,43) and subsequently confirmed by other studies(44–46) as a novel member of the TNF-receptor superfamily. Other synonyms for this factor are TNF receptor–related molecule 1 (TR1)(44,45) and follicular dendritic cell receptor 1 (FDCR-1).(46) OPG/OCIF is synthesized as a propeptide (401 amino acids for the human, mouse, and rat forms), of which the signal peptide (21 amino acids) is cleaved, thus generating the mature peptide (380 amino acids).(8,9,43,44) In contrast to all other TNFR superfamily members, OPG/OCIF lacks transmembrane and cytoplasmic domains and is secreted as a soluble protein.(8,9,44,45)

The N terminus contains four domains (D1–D4) with cysteine-rich motifs(8,9,44,45) involved in the formation of “tethered loops”(44) and is most closely related to TNFR-2 and CD40.(8) The C terminus contains two death domain–homologous (DDH) tandem regions (D5 and D6) that are closely related to Fas and TNFR-1,(9) and a domain (D7) with a heparin-binding site and a cysteine residue (position 400) required for homodimer formation.(47) The integrity of all four cysteine-rich motifs (D1–D4) appears to be required for the biological activity of OPG/OCIF.(8,47) Furthermore, when fused to the transmembrane region of Fas, the C terminus (D5–D7) can mediate cytotoxicity directly.(47) OPG/OCIF is synthesized as a monomer (55–62 kDa), assembled, and then secreted as a disulfide-linked homodimeric glycoprotein with 4–5 potential N-glycosylation sites (110–120 kDa).(8,43,45) Although the physicochemical properties of the OPG/OCIF monomer and homodimer are similar, the homodimer has a higher heparin-binding ability and a higher hypocalcemic potency.(43,48)

The human OPG/OCIF gene has been mapped to chromosome 8q23–24(8,44) and contains five exons distributed over 29 kilobases (kb).(47,49) Of interest, exon 4, which encodes most of the first DDH region, is a partial duplication of exon 5.(47) Three major OPG/OCIF mRNA species exist: there are one abundant 2.2- to 3.0-kb species and two minor species of 4.2–4.4 kb and 6.5–6.6 kb, respectively.(8,9,44,45) The two larger species are splice variants that contain either the entire intron 2 (6.5- to 6.6-kb species) or the 3′ half of intron 2 (4.2- to 4.4-kb species).(47)

Expression and regulation

Compared with that of OPG-L/ODF, steady state levels of OPG/OCIF mRNA are higher and have a wider tissue distribution, that is not restricted to bone or immune tissues.(8,9,44,45) High levels of OPG/OCIF mRNA have been detected in lung, heart, kidney, liver, stomach, intestine, skin, brain and spinal cord, thyroid gland, and bone.(8,9,44,45) During mouse embryogenesis (day 15), OPG/OCIF mRNA was localized to the cartilaginous parts of developing bone, the aorta, the gastrointestinal tract, and the skin by in situ hybridization.(8) OPG/OCIF mRNA levels have been subsequently detected in a variety of osteoblastic lineage cells, including marrow stromal cell lines,(9,44,50) osteoblastic cell lines,(17,51,52) and osteosarcoma cell lines MG63.(17,44,45,50) In addition, high OPG/OCIF mRNA levels have also been detected in endothelial cells and aortic smooth muscle cells,(44,45) fibroblastic cells,(9,44,45) ovarian (CAOV-3)(45) and breast cancer cell lines (MCF7),(44) and monocytic, dendritic, and B lymphocytic cell lines.(44,45)

Levels of OPG/OCIF mRNA and protein in osteoblastic lineage cells are increased by the cytokines IL-1α, IL-1β,(51,53) TNF-α, and TNF-β,(51,54) by the osteoblast-differentiating factors 1α,25-(OH)2D3 and bone morphogenetic protein 2(51) and by the antiresorptive agents estrogen(52) and TGF-β.(18,55) OPG/OCIF mRNA levels of murine marrow stromal cell lines are increased by treatment with calcium chloride.(9) By contrast, OPG/OCIF mRNA and protein levels are decreased by glucocorticoids,(17,50) PGE2,(56) and by the pure estrogen receptor antagonist ICI 182,780.(52)

Biological effects

OPG/OCIF acts as a soluble secreted receptor for OPG-L/ODF that prevents it from binding to and activating ODAR/RANK on the osteoclast surface. Thus, the biological effects of OPG/OCIF on bone cells are the opposite of that of OPG-L/ODF, including inhibition of the terminal stages of osteoclast differentiation,(8,9,43,45,57–60) suppression of the activation of mature osteoclasts,(10,22,23,45,61) and induction of apoptosis(49) (Fig. 1). By using a spleen cell/bone marrow stromal cell coculture system, Simonet et al.(8) have shown that the human recombinant OPG/OCIF dimer inhibited osteoclast differentiation by 50% at a concentration of 1 ng/ml and completely suppressed osteoclast differentiation at concentrations of 10 to 100 ng/ml as assessed by TRAP activity and cytochemistry. By using a monomeric and dimeric OPG/OCIF preparation derived from conditioned medium of fetal pulmonary fibroblasts, Tsuda et al.(43) have reported that osteoclast differentiation was inhibited at concentrations from 1 to 40 ng/ml with a half-maximal effect at 4–6 ng/ml. In an experiment assessing the sensitivity of osteoclastogenesis to OPG/OCIF in vitro, when OPG/OCIF was added or withdrawn over a period of 11 days, continuous exposure from days 5 through 11 was required for osteoclast inhibition, whereas withdrawal after day 3 or intermittent administration on days 7 and 8 had no effect.(49) Treatment with OPG/OCIF also inhibited the osteoclastogenesis induced by treatment with 1α,25(OH)2D3, PGE2, PTH, IL-1, or IL-11.(43,45,60)

In addition to blocking osteoclast formation, OPG/OCIF also inhibits osteoclastic pit formation of mature osteoclasts.(21,45,61) It also antagonizes the induction of bone resorption by 1α,25(OH)2D3, PGE2, PTH, and IL-1α(26,45) as well as OPG-L/ODF.(21) Compared with its effect on inhibition of osteoclast differentiation, however, higher concentrations of OPG/OCIF (50–1000 ng/ml) are required to inhibit osteoclast activation.(26,45,61) Whereas OPG/OCIF was initially reported to have no effect on various nonosteoclastic cells and cell lines,(45) more recently high levels OPG/OCIF (200 ng/ml) have been reported to specifically increase human foreskin fibroblast proliferation(45) and, because OPG/OCIF also acts as a soluble receptor for TRAIL, to inhibit TRAIL-induced apoptosis of the Jurkat T-cell line.(59)

The impressive antiresorptive effects of OPG/OCIF have also been shown in several in vivo studies. Transgenic mice overexpressing OPG/OCIF have severe osteopetrosis with narrowing of the bone marrow cavities, resulting in splenomegaly due to compensatory extramedullary hematopoiesis.(8) Histological analysis revealed a systemic increase in mineralized trabecular bone associated with a marked decrease in the number of trabecular osteoclasts.(8) In contrast to OPG-L/ODF-knockout mice,(29) transgenic mice overexpressing OPG/OCIF lacked defects of tooth eruption, lymphocyte development, or lymph node organogenesis.(8) A possible explanation for the differences in these phenotypes may be that the OPG-L/ODF gene was completely inactivated during embryogenesis, whereas the OPG/OCIF transgene was activated only after birth, thus permitting OPG-L/ODF effects during embryogenesis.

Conversely, targeted ablation of OPG/OCIF resulted in mice with severe osteoporosis,(62,63) destruction of the femoral growth plates, and multiple fractures during the 1st two months of life, and increased postnatal mortality.(62,63) Adult OPG/OCIF-deficient mice had serial vertebral compression fractures and collapsed femur epiphyses.(62) The bone mineral density of OPG/OCIF-deficient mice at 2 months of age was 19% (cortical bone) to 45% (trabecular bone) lower than that of the wild-type littermates.(62) By 3 months of age, strength and stiffness of bone in OPG/OCIF-deficient male mice were reduced by more than 50%.(63) Histological analysis of the bones of OPG/OCIF-deficient mice revealed loss of cancellous and cortical bone in the first and fourth weeks of life, respectively,(62) with near-total lack of cancellous bone after 2 months of life.(62,63) This was associated with markedly increased bone turnover: there was increased osteoclastic and osteoblastic activity as assessed by bone histomorphometry, and serum alkaline phosphatase activity was four times higher in OPG/OCIF-deficient mice than in wild-type mice.(62) Of interest, OPG/OCIF-deficient mice also had profound calcification of the large arteries by 2 months of age that progressed to marked medial calcification of the aorta and the renal arteries, profound intimal and medial proliferation, and partial aortic dissection by 4 months.(62) Because OPG/OCIF is expressed in the media of large arteries in the wild-type mice,(8) it may play a protective role in vascular biology. Interestingly, the degrees of vascular calcification and osteoporosis are correlated in aging women.(64)

PARACRINE REGULATION OF OSTEOCLASTOGENESIS: INTEGRATION OF MECHANISMS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. OSTEOPROTEGERIN LIGAND/OSTEOCLAST DIFFERENTIATION FACTOR
  5. THE RECEPTOR FOR OPG-L/ODF
  6. OPG/OCIF
  7. PARACRINE REGULATION OF OSTEOCLASTOGENESIS: INTEGRATION OF MECHANISMS
  8. RELEVANCE TO METABOLIC BONE DISORDERS
  9. POTENTIAL THERAPEUTIC USES OF OPG
  10. Acknowledgements
  11. REFERENCES

As mentioned earlier, deletion of OPG-L/ODF(29) or its target transcription factor, NF-κB,(35) and overexpression of OPG/OCIF(8) or soluble ODAR/RANK(32) result in marked osteopetrosis, whereas deletion of OPG/OCIF(62,63) or exogenous administration of OPG-L(10) results in severe osteoporosis and hypercalcemia. Because manipulation of the components of the OPG-L/OPG system can produce both extremes of skeletal phenotype and because OPG-L, in the presence of M-CSF, is necessary and sufficient for osteoclastogenesis, the effects of many calcitropic cytokines and hormones on bone resorption may be mediated through regulation of the production of OPG-L/ODF and/or its endogenous receptor antagonist, OPG/OCIF. The differential regulation of OPG-L/ODF and OPG/OCIF by various proresorptive and antiresorptive agents (Table 2) suggests that their effect may converge at the level of OPG-L/ODF and OPG/OCIF, which then functions as the final effector system to modulate differentiation, activation, and apoptosis of osteoclasts (Fig. 2). For example, the stimulation of OPG-L/ODF gene expression by IL-1β, IL-11, TNF-α, PTH, and PGE2(11,17) and the inhibition of OPG/OCIF gene expression by PGE2(56) may mediate the proresorptive effects of these factors. This “convergence hypothesis” is consistent with the finding that OPG/OCIF or OPG-L/ODF-neutralizing antibodies block the increase of bone resorption in a murine long bone culture system stimulated by 1α,25-(OH)2D3, IL-1α, PTH, and PGE2, all factors that stimulate OPG-L/ODF production.(26) By contrast, TGF-β, a known antiresorptive growth factor,(65,66) may act through suppression of OPG-L/ODF(18) and concurrent stimulation of OPG/OCIF (Fig. 2).(18,55) The convergence of changes in multiple upstream hormones and cytokines to regulation of only two downstream cytokines (OPG/OCIF or OPG-L/ODF [with a permissive effect of M-CSF]) would represent a regulatory mechanism for osteoclastogenesis of great efficiency and elegance. Similar to the IL-1 system, in which the stimulatory effects of IL-1α and IL-1β are counterbalanced by the naturally occurring endogenous receptor antagonist IL-1 receptor antagonist, both of which are differentially regulated in various systems,(67) modulation of OPG-L/ODF and its soluble receptor antagonist OPG/OCIF allows a fine-tuned, two-way regulation of osteoclastogenesis. The interaction among OPG/OCIF, OPG-L/ODF, and ODAR/RANK as final effectors of osteoclastogenesis are shown in Fig. 3.

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Figure FIG. 2. “Convergence” hypothesis” for the regulation of osteoclast functions by cytokines. This hypothesis proposes two levels of regulation of osteoclast functions. A variety of “upstream” cytokines and hormones alter the pool size of active osteoclasts by converging at the level of OPG-L/ODF and OPG/OCIF. These two “downstream” factors serve as the final effectors for osteoclastogenesis and also affect osteoclast activation and osteoclast apoptosis. At steady state, there is a “balance” of levels of OPG-L/ODF and OPG/OCIF that maintain a pool size of active osteoclasts that supports normal levels of bone resorption. When a change in one or more upstream factors tilts the balance toward a functional excess of OPG-L/ODF, the pool size of active osteoclasts increases; when the balance tilts toward a functional excess of OPG/OCIF, the pool size decreases. 17β-E2, 17β-estradiol; PGE2, prostaglandin E2; PTH, parathyroid hormone; TGF-β, transforming growth factor β.

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thumbnail image

Figure FIG. 3. Regulation of osteoclastogenesis by the interaction of OPG-L/ODF, OPG/OCIF, and ODAR/RANK in the bone marrow microenvironment. Stromal cells and osteoblasts produce both M-CSF and OPG-L/ODF. In the presence of permissive concentrations of cellular or soluble M-CSF, binding of OPG-L/ODF to its receptor ODAR/RANK on osteoclast precursor cells results in their differentiation and activation. OPG-L/ODF can bind to the receptor in a soluble (sOPG-L/ODF) or cell-bound (cOPG-L/ODF) form, the latter of which requires cell-to-cell contact (broken line). In contrast, OPG/OCIF is produced only in a soluble form and, after secretion, competes with ODAR/RANK for binding of OPG-L/ODF by neutralizing OPG-L/ODF.

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While a number of pro-resorptive cytokines have been shown to stimulate OPG-L/ODF production, the data regarding IL-6 effects on the OPG-L/ODF and OPG/OCIF system are somewhat conflicting. Several studies have found that, in contrast to IL-1 or TNF-α, IL-6 did not affect OPG/OCIF production.(51,53,54) In addition, IL-6 (with or without its soluble receptor) did not have any effects on OPG-L/ODF mRNA levels in human marrow stromal cells or MG-63 cells.(17) Moreover, a recent finding indicates that IL-6 may increase osteoclastogenesis through an alternative pathway that is independent of the OPG-L/NF-κB signaling pathway.(68) OPG-L/ODF, M-CSF, IL-1β, and TNF-α did not induce osteoclastogenesis in cells derived from mice that were deficient in both NF-κB1 and NF-κB2.(68) However, IL-6, in the presence of its soluble receptor, induced osteoclast formation in these double-knockout mice, albeit to only 6% of that induced by OPG-L/ODF in wild type mice.(68) In contrast to these findings, O'Brien et al.(69) recently reported that IL-6 did up-regulate OPG-L/ODF mRNA levels in a murine stromal/osteoblastic cell line (UAMS-32) and that this up-regulation was essential for the ability of these cells to increase osteoclastogenesis in response to IL-6. Finally, it is possible that some of the bone-resorbing effects of IL-6 may result from enhanced osteoclastic activity. Adebanjo et al.(70) have recently shown that IL-6, which is stimulated by increasing calcium concentrations generated locally in areas of bone resorption, may attenuate calcium sensing and thus increase bone resorption through a direct effect on osteoclast activity.

RELEVANCE TO METABOLIC BONE DISORDERS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. OSTEOPROTEGERIN LIGAND/OSTEOCLAST DIFFERENTIATION FACTOR
  5. THE RECEPTOR FOR OPG-L/ODF
  6. OPG/OCIF
  7. PARACRINE REGULATION OF OSTEOCLASTOGENESIS: INTEGRATION OF MECHANISMS
  8. RELEVANCE TO METABOLIC BONE DISORDERS
  9. POTENTIAL THERAPEUTIC USES OF OPG
  10. Acknowledgements
  11. REFERENCES

Bone loss due to estrogen deficiency

Estrogen deficiency is the main cause of bone loss in postmenopausal women and may also contribute to bone loss in aging men.(71) Estrogen acts directly on both osteoblasts and osteoclasts through high-affinity estrogen receptors.(72) However, the paracrine mediation of the estrogen effects are not clearly defined. Although estrogen deficiency increases both the formation and activation of osteoclasts, the former process probably contributes more to the increased bone loss than does the latter.(73) There is evidence that the increase in osteoclastogenesis induced by estrogen deficiency is related to increased production of multiple proresorptive cytokines, including IL-1, TNF-α, IL-6, M-CSF, and PGE2,(74–78) and to impaired production of the antiresorptive cytokine IL-1 receptor antagonist.(79) These changes are reversed by estrogen administration.(74–79) Estrogen deficiency also increases activation(80) and decreases apoptosis(81) of mature osteoclasts, and these effects are also reversed by estrogen.

Interestingly, these effects of estrogen appear to be mediated, at least in part, by TGF-β(81): estrogen has been shown to increase both TGF-β production by osteoblasts(82) and by osteoclasts,(83) and TGF-β stimulates the production of OPG/OCIF.(18,55) Moreover, estrogen also increases the production of OPG/OCIF by mature osteoblasts,(52) and exogenous administration of OPG/OCIF to oophorectomized rats completely prevented bone loss,(8) suggesting that the OPG-L/OPG system may serve as an important paracrine mediator of the antiresorptive effects of estrogen.

Recently, Yano et al.(84) reported a threefold increase in serum OPG/OCIF levels with aging in both women and men and a small but significant increase in serum OPG/OCIF levels in osteoporotic postmenopausal women compared with normal postmenopausal women. They suggested that these changes represent a compensatory response to the increase in bone resorption. In contrast, both Arrighi et al.(85) and our group (B. L. Riggs, L. J. Melton, C. R. Dunstan, and S. Khosla, unpublished data) were not able to find major increases in serum levels of OPG/OCIF with age in large cohorts of women and men. Clearly, more studies are needed to examine these interesting questions further.

Bone loss due to glucocorticoid excess

The rapid development of severe osteoporosis is a frequent complication of systemic glucocorticoid therapy and one of the major limitations to its use.(86,87) The cause of bone loss is both an increase in (or at least sustained) bone resorption and a decrease in bone formation.(86,87) However, although various in vitro(88–90) and in vivo(91–93) data suggest an increase of osteoclastogenesis and bone resorption, the molecular mechanism(s) underlying this effect are unclear. Because glucocorticoids differentially regulate the OPG-L/ODF system by decreasing OPG/OCIF by up to 90%(16,50) and increasing OPG-L/ODF by up to fourfold,(11,16) thus markedly increasing the OPG-L/ODG ratio, OPG/OCIF and OPG-L/ODF are strong candidate cytokines for the proresorptive effects of glucocorticoids on bone. In support of this, OPG serum concentrations decreased significantly in patients undergoing systemic glucocorticoid therapy from 0.30 ± 0.03 ng/ml at baseline to 0.23 ± 0.02 ng/ml after 4 weeks.(94)

POTENTIAL THERAPEUTIC USES OF OPG

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. OSTEOPROTEGERIN LIGAND/OSTEOCLAST DIFFERENTIATION FACTOR
  5. THE RECEPTOR FOR OPG-L/ODF
  6. OPG/OCIF
  7. PARACRINE REGULATION OF OSTEOCLASTOGENESIS: INTEGRATION OF MECHANISMS
  8. RELEVANCE TO METABOLIC BONE DISORDERS
  9. POTENTIAL THERAPEUTIC USES OF OPG
  10. Acknowledgements
  11. REFERENCES

If our “convergence hypothesis” is correct that the pool size of active osteoclasts is mainly determined by the ratio of OPG-L/ODF to OPG/OCIF in the bone and bone marrow microenvironment, administration of OPG/OCIF would be rational treatment for metabolic bone diseases characterized by increased bone resorption. The therapeutic potential of OPG/OCIF is supported by findings of several animal studies. Subcutaneous injections of recombinant OPG/OCIF (10 mg/kg/day) for 7 days (leading to an OPG/OCIF serum steady-state level of 320 ± 176 ng/ml) increased trabecular bone mass at the proximal tibia in 4-week-old mice threefold.(8) Moreover, a similar treatment regimen with OPG/OCIF (5 mg/kg/d) for 2 weeks was also able to decrease osteoclastic bone resorption, to prevent bone loss after ovariectomy, and even to increase bone volume.(8) Furthermore, OPG/OCIF, by virtue of rapidly inhibiting osteoclast activity may have therapeutic potential in the treatment of hypercalcemia: hypocalcemic effects of OPG/OCIF have been reported in animal models of enhanced osteoclastic bone resorption, including during longitudinal bone growth and after IL-1 challenge,(95) and humoral hypercalcemia of malignancy.(95,96) In addition, OPG/OCIF was also found to lower serum calcium levels in normal animals,(96) in hypercalcemic animals after the administration of OPG-L/ODF,(8) or in animals with hypercalcemia generated by thyroparathyroidectomy and subsequent treatment with PTH and 1α,25-(OH)2D3.(27) These observations indicate the therapeutic potential of recombinant OPG/OCIF to prevent or to treat enhanced bone resorption and bone loss, to increase bone mass, and to control severe hypercalcemia.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. OSTEOPROTEGERIN LIGAND/OSTEOCLAST DIFFERENTIATION FACTOR
  5. THE RECEPTOR FOR OPG-L/ODF
  6. OPG/OCIF
  7. PARACRINE REGULATION OF OSTEOCLASTOGENESIS: INTEGRATION OF MECHANISMS
  8. RELEVANCE TO METABOLIC BONE DISORDERS
  9. POTENTIAL THERAPEUTIC USES OF OPG
  10. Acknowledgements
  11. REFERENCES

We thank Dr. Thomas C. Spelsberg for his helpful comments. The authors' research was supported by grant AG-04875 from the National Institutes of Health (to B.L.R. and S.K.) and a postdoctoral fellowship grant from the Deutsche Forschungsgemeinschaft (Ho 1875/1–1) and the Mayo Foundation (to L.C.H.).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. OSTEOPROTEGERIN LIGAND/OSTEOCLAST DIFFERENTIATION FACTOR
  5. THE RECEPTOR FOR OPG-L/ODF
  6. OPG/OCIF
  7. PARACRINE REGULATION OF OSTEOCLASTOGENESIS: INTEGRATION OF MECHANISMS
  8. RELEVANCE TO METABOLIC BONE DISORDERS
  9. POTENTIAL THERAPEUTIC USES OF OPG
  10. Acknowledgements
  11. REFERENCES
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