Osteoprotegerin

Osteoprotegerin William J. Boyle* Department of Cell Biology, Amgen, Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA * corresponding author tel: 805-447-4304, fax: 805-447-1982, e-mail: [email protected] DOI: 10.1006/rwcy.2000.16009

SUMMARY Osteoprotegerin (OPG) is a new member of the TNFR superfamily that functions solely as a secreted cytokine antagonist or decoy receptor. The structure and function of OPG is highly conserved during evolution, involves the binding to, and neutralization of RANKL during the regulation of bone metabolism. OPG is secreted by expressing cells, and can bind tightly to extracellular matrix proteins via a C-terminal heparin-binding domain. Overexpression of OPG, or systemic administration of recombinant protein, blocks osteoclast differentiation and activation in mice, rats, primates, and humans. The resulting effect is to negatively regulate bone resorption, which leads to increases in bone mass. Loss of OPG results in early onset osteoporosis in mice, leading to brittle bone structure and accumulation of long bone fractures. OPG recombinant protein can block the pathological bone loss that occurs following estrogen loss, during adjuvant-induced arthritis, and during the growth of lytic tumors, indicating a possible use in the treatment of osteopenic disorders characterized by increased osteoclast activity.

The primary structure of OPG suggests that it can act in the extracellular milieu as a receptor-like protein that binds to, and regulates, the activity of a TNFrelated ligand (see Suda et al., 1999 for review). The term `osteoprotegerin' simply refers to a protein that protects or guards against the destruction of bone. Based on in vitro and in vivo analysis, OPG has been found to negatively regulate osteoclastogenesis and osteoclast activation, and in doing so, to control bone remodeling and density. Osteoprotegerin was first identified as a novel expressed sequence tag (EST) from a cDNA library prepared from fetal rat intestine, independent of any perceived biological function, i.e. via genomics (Adams et al., 1992). Computational analysis of this sequence revealed that it encoded a homolog of the TNFR superfamily of receptors. Using this sequence information, full-length rat, mouse, and human cDNA clones were isolated, and the amino acid sequence of its protein product was deduced. OPG was also purified from the conditioned media produced by human fibroblasts as a factor that inhibited osteoclast development from hematopoietic precursors (Tsuda et al., 1997).

BACKGROUND

Alternative names

Discovery Osteoprotegerin (OPG) is a recently described protein that appears to be a major factor affecting bone metabolism (Simonet et al., 1997). It is a member of the tumor necrosis factor receptor superfamily, with special properties. OPG is a secreted protein, and not a traditional surface membrane-associated receptor that transmits signals regulating cell functions. In this regard it is unlike most other TNFR family members.

OCIF, osteoclastogenesis inhibitory factor (Yasuda et al., 1998a) TR1, TNF receptor-like protein 1 (Kwon et al., 1998) FDCR-1, follicular dendritic cell receptor 1 (Yun et al., 1998)

Structure OPG is a secreted TNFR-related protein with two functional domains (Figure 3). The N-terminal half

1700 William J. Boyle of OPG contains four tandem cysteine-rich repeat sequences that are characteristic of the ligand-binding domains of all members of this family. The Cterminal half of the protein has no distinct homologies to any known proteins, and is involved in OPG homodimerization following biosynthesis. The C-terminal domain binds to heparin, and may be involved in localization to extracellular matrix.

Main activities and pathophysiological roles OPG plays a central role in regulating skeletal metabolism by inhibiting differentiation and activation of the osteoclast. Bone remodeling and homeostasis is an essential function that regulates skeletal integrity throughout adult life in higher vertebrates and mammals (Suda et al., 1992; Roodman, 1996). The maintenance of skeletal mass is controlled by the activities of specialized cells within the bone that have seemingly antagonistic activities: bone synthesis and bone resorption. Osteoblastic cells of mesenchymal origin synthesize and deposit bone matrix and increase bone mass. Osteoclastic cells are large, multinucleated phagocytes of hematopoietic origin that resorb both mature and newly synthesized bone upon activation. Bone synthesis and resorption processes are highly coordinated, and are controlled by osteotropic and calciotropic hormones during physiological and pathological conditions (Rodan and Martin, 1981; Martin and Udagawa, 1998). Osteoclast recruitment and activation can be accelerated in certain disease processes, leading to inappropriate increases in bone resorption, and subsequently a net loss of bone mass. Increased osteoclast activity is associated with bone loss in several disorders, including primary osteoporosis, Paget's disease of the bone, hypercalcemia of malignancy, and rheumatoid arthritis. Understanding the biology of the osteoclast is an important field of study, and carries with it implications that may lead to the development of effective therapies to treat bone loss in humans. The ability of OPG to regulate bone resorption and remodeling in vivo implicates the protein as a key determinant in the regulating bone mass and calcium metabolism. OPG does not act alone, it effects a pathway of hematopoietic cell development that is dependent on a key ligand/receptor interaction found to be essential for osteoclast maturation and activation. OPG has been useful as a reagent to probe the nature of this regulatory pathway, and combined biochemical, cell biological, and molecular

genetic analysis in mice has provided a clear mechanism-based view of how osteoclastogenesis is regulated. Osteoclasts are specialized macrophage cells that develop from hematopoietic precursors in the bone marrow and spleen. They stem from a common monocyte/macrophage precursor that can give rise to several mature macrophage cell types, including avelolar macrophages and hepatic KuÈpffer cells. At the time OPG was first identified and characterized, little was actually known about the various steps involved in the commitment to the osteoclast lineage, and the processes that control osteoclast maturation (osteoclastogenesis). However, using in vitro culture systems, mature osteoclasts could be formed from bone marrow and spleen cells (Udagawa et al., 1989). The polypeptide factor M-CSF (CSF-1), along with stromal cells, vitamin D3, and dexamethasone are required for osteoclastogenesis to occur in this in vitro systems. A vitamin D3-dependent bone marrow and stromal cell co-culture system was used to determine the effects of OPG on osteoclast development (Udagawa et al., 1989; Lacey et al., 1995). This assay system was used to purify the native human OPG polypeptide to homogeneity from conditioned media produced by a normal human diploid lung fibroblast cell line (Tsuda et al., 1997). The protein product that was purified included both homodimeric and monomeric OPG polypeptide chains, similar to the expression product characterized in the circulation of OPG transgenic mice (Simonet et al., 1997). Native OPG and recombinant OPG protein has been shown to block in vitro osteoclastogenesis in a dose-dependent manner, with a half-maximal effective concentration in the range of 1±2 ng/mL. OPG blocks the formation of large multinucleated osteoclasts that express tartrate-resistant acid phosphatase (TRAP) activity, the calcitonin receptor, and the integrin v 3, all markers of the mature osteoclast (Figure 5). Only the N-terminal half of the protein, which resembles a ligand-binding domain, is required for biological activity in this in vitro system (Simonet et al., 1997; Yamaguchi et al., 1998).

GENE

Accession numbers Rat OPG cDNA: U94331 Murine OPG cDNA: U94331, E15271 Human OPG cDNA: U94332, AB013898 Mouse OPG gene: AB013899S

Osteoprotegerin 1701

Sequence

Description of protein

See Figure 1.

The long open reading frame of the OPG OPG/OCIF encodes a 401 residue polypeptide with two functional regions. The N-terminal half of OPG (residues 22± 185) consists of four tandem cysteine-rich repeat sequences, and bears striking homology to the ligandbinding domain of all TNFR family members (Smith et al., 1994). The C-terminal half of the protein (residues 186±401) bears no obvious homologies to other known proteins, but appears to contain minimal sequence identity to a portion of the death domain motif found on certain intracellular proteins (Simonet et al., 1997; Yamaguchi et al., 1998). This region is involved in OPG homodimerization and extracellular matrix binding. At the extreme N-terminus of the protein lies a functional 21 amino acid hydrophobic

PROTEIN

Accession numbers Rat OPG protein: AAB53707 Murine OPG protein: AAB53708, AB013903 Human OPG protein: AAB53709, AB002146

Sequence See Figure 2.

Figure 1 Nucleotide sequence for OPG. Human OPG cDNA Sequence: 1 GTATATATAA CGTGATGAGC 61 CGYCTCCAAG CCCCTGAGGT 121 CGTGTTTCTG GACATCTCCA 181 TCATTATGAC GAAGAAACCT 241 CCTAAAACAA CACTGTACAG 301 CTACACAGAC AGCTGGCACA 361 GCTGCAGTAC GTCAAGCAGG 421 AGGGCGCTAC CTTGAGATAG 481 AGTGGTGCAA GCTGGAACCC 541 CTTCTCAAAT GAGACGTCAT 601 TGGTCTCCTG CTAACTCAGA 661 TGAATCAACT CAAAAATGTG 721 TGCTGTTCCT ACAAAGTTTA 781 CACCAAAGTA AACGCAGAGA 841 GACTTTCCAG CTGCTGAAGT 901 GATCATCCAA GATATTGACC 961 CCTCACCTTC GAGCAGCTTC 1021 AGAAGACATT GAAAAAACAA 1081 CAGTTTGTGG CGAATAAAAA 1141 AAAGCACTCA AAGACGTACC 1201 CAGGTTCCTT CACAGCTTCA 1261 AGGTAACCAG GTCCAATCAG 1321 GTTTCCTCAC AATTGGCGAG

GTACGGGTGC TTCCGGGGAC TTAAGTGGAC CTCATCAGCT CAAAGTGGAA CCAGTGACGA AGTGCAATCG AGTTCTGCTT CAGAGCGAAA CTAAAGCACC AAGGAAATGC GAATAGATGT CGCCTAACTG GTGTAGAGAG TATGGAAACA TCTGTGAAAA GTAGCTTGAT TAAAGGCATG ATGGCGACCA ACTTTCCCAA CAATGTACAA TAAAAATAAG ATCCCATGGA

GGAGACGCAC CACAATGAAC CACCCAGGAA GTTGTGTGAC GACCGTGTGC GTGTCTATAC CACCCACAAC GAAACATAGG TACAGTTTGC CTGTAGAAAA AACACACGAC TACCCTGTGT GCTTAGTGTC GATAAAACGG TCAAAACAAA CAGCGTGCAG GGAAAGCTTA CAAACCCAGT AGACACCTTG AACTGTCACT ATTGTATCAG CTGCTTATAA TGATAA

CGGAGCGCTC AAGTTGCTGT ACGTTTCCTC AAATGTCCTC GCCCCTTGCC TGCAGCCCCG CGCGTGTGCG AGCTGCCCTC AAAAGATGTC CACACAAATT AACATATGTT GAGGAGGCAT TTGGTAGACA CAACACAGCT GCCCAAGATA CGGCACATTG CCGGGAAAGA GACCAGATCC AAGGGCCTAA CAGAGTCTAA AAGTTATTTT CTGGAAATGG

GCCCAGCCGC GCTGCGCGCT CAAAGTACCT CTGGTACCTA CTGACCACTA TGTGCAAGGA AATGCAAGGA CTGGATTTGG CAGATGGGTT GCAGTGTCTT CCGGAAACAG TCTTCAGGTT ATTTGCCTGG CACAAGAACA TAGTCAAGAA GACATGCTAA AAGTGGGAGC TGAAGCTGCT TGCACGCACT AGAAGACCAT TAGAAATGAT CCATTGAGCT

Figure 2 Amino acid sequence for OPG. Human OPG protein 1 MNKLLCCALV 61 CAPCPDHYY 121 HRSCPPGFGV 181 HDNICSGNSE 241 KRQHSSQEQT 301 SLPGKKVGAE 361 VTQSLKKTIR

sequence: FLDISIKWTT TDSWHTSDEC VQAGTPERNT STQKCGIDVT FQLLKLWKHQ DIEKTIKACK FLHSFTMYKL

QETFPPKYLH LYCSPVCKEL VCKRCPDGFF LCEEAFFRFA NKAQDIVKKI PSDQILKLLS YQKLFLEMIG

YDEETSHQLL QYVKQECNRT SNETSSKAPC VPTKFTPNWL IQDIDLCENS LWRIKNGDQD NQVQSVKISC

CDKCPPGTYL HNRVCECKEG RKHTNCSVFG SVLVDNLPGT VQRHIGHANL TLKGLMHALK L

KQHCTAKWKT RYLEIEFCLK LLLTQKGNAT KVNAESVERI TFEQLRSLME HSKTYHFPKT

1702 William J. Boyle signal peptide that directs secretion and is cleaved during biosynthesis. Further analysis of the OPG amino acid sequence failed to reveal potential any hydrophobic transmembrane-spanning domain such as that seen in other members of the TNFR family, and was an early indication that it might be a secreted protein. Pulsechase studies in mammalian cell lines that overexpress the murine OPG cDNA revealed that it is indeed a secreted protein that is proteolytically processed after synthesis, modified by glycosylation, and exported into the conditioned media. Analysis of the biosynthetic product under nonreducing conditions also reveals that the primary OPG polypeptide self associates during secretion, and is secreted from the cell both as a disulfide-linked dimer, and as a monomer (Simonet et al., 1997; Tsuda et al., 1997). Structural analysis of the C-terminal region indicates that cysteine residue at position 400 is critical for the formation of secreted homodimer (Yamaguchi et al., 1998). Dimerization and oligomerization of TNFR-related proteins are associated with high affinity for ligand. Formation of a secreted dimer is a unique property for members of the TNFR superfamily, and suggests that OPG is biosynthesized as a high-affinity cytokine antagonist. Structure and function analyses of OPG reveals that the protein region required for biological activity resides within the N-terminal 185 amino acids, which harbors the four tandem cysteine-rich repeat sequences (Figure 3). All four domains are required for biological activity, as well as each of the 18 individual cysteine residues that stabilize the tertiary structure of these motifs. This region of TNFRrelated proteins comprises the ligand-binding domain of members to this family, implying that OPG activity is mediated via binding to a TNF-related protein. The C-terminal domain of the protein can be deleted, and OPG molecules retain biological activity in vitro and in vivo, further suggesting that OPG bioactivity involves binding to a TNF-related cytokine.

Relevant homologies and species differences The rat, mouse, and human OPG cDNAs have been cloned and sequenced, and their protein products compared. OPG sequences are highly conserved during evolution at the DNA and protein level. The rat and mouse proteins are about 94% identical, whereas the mouse and human proteins are about 89% identical, without sequence gaps. All of the cysteine residues located in the full-length mature

Figure 3 Structure of rat OPG mRNA and protein. The rat OPG mRNA is a 2.4 kb transcript and is represented by the thin line. The 401 amino acid long open reading frame encoding the rat OPG product is represented by the coded box. The first 21 amino acids (black) indicate the position of the signal peptide sequence. The N-terminal half of OPG contains four tandem cysteine-rich repeat sequences (shaped ellipses). The C-terminal dimerization domain is indicated by a gray box. The coding frame begins at a methionine codon (AUG), and terminates at a stop codon (TAG) following leucine 401. C185 represents the last cysteine residue of the TNFR homology domain. The mouse and human OPG cDNA sequences also encode 401 amino acid polypeptides that are approximately 89± 94% identical to the rat protein shown here. Below is a illustration of the mature secreted form of the OPG homodimer, beginning at amino acid reisdue 22 and ending at 401. The last disulfide residue of the cysteinerich domain IV is labeled as Cys185.

proteins (residues 22±401) are conserved and in identical positions.

Affinity for ligand(s) The biological activity of OPG strongly suggests that it negatively regulates a cytokine that stimulates osteoclast maturation and activation. It had been known for many years that osteoblastic cells could be stimulated by calciotropic agents to produce a factor that stimulates osteoclast development and activation (Suda et al., 1999). The ligand for OPG was a likely candidate for this factor, but had not yet been identified and its biological activities remained elusive. Using OPG as a probe, a putative OPG ligand was expression cloned from cDNA libraries made from osteoblastic stromal cells induced with vitamin D3 (Yasuda et al., 1998b), or from the

Osteoprotegerin 1703 murine myelomonocytic cell line 32D (Lacey et al., 1998). Both clones encoded an identical 316 amino acid mouse protein that was termed osteoclast differentiation factor (ODF) and OPG ligand (OPGL) (see RANKL). The protein product was predicted to be a type II transmembrane surface protein with clear structural motifs found in TNF family members. OPG ligand (RANKL) can be cleaved from the surface of expressing cells (Lacey et al., 1998) and released in a soluble, biologically active form. It is not yet clear whether the soluble form of OPG ligand plays a role in regulating osteoclast function(s) in vivo, although recombinant forms of soluble of the protein have potent bioactivity when administered to animals (Lacey et al., 1998). The cDNA encoding ODF/ OPGL had previously been isolated by two different approaches that suggested a role for this cytokine in regulation of immune responses. The first report of this sequence was the differential cloning of a T cell protein induced by calcineurin-regulated transcription factors, and named TRANCE (TNF related activation-induced cytokine) (Wong et al., 1998). It was subsequently also identified as RANKL, a ligand for a novel TNFR-related protein RANK (receptor activator of NFB), a dendritic cell surface protein (Anderson et al., 1997). Activated T cells and some T cell lines express TRANCE/RANKL, suggesting a role in costimulatory processes during antigen processing by regulating dendritic cell survival (Wong et al., 1997; Anderson et al., 1997). OPG has also been shown to bind to the TNFrelated cytotoxin TRAIL (Emery et al., 1998). The coaddition of TRAIL and OPG during in vitro osteoclast-forming assays indicates that recombinant TRAIL can block OPG activity. This suggests that TRAIL may regulate OPG function during bone metabolism in vivo. Administration of recombinant TRAIL protein into mice at relatively high doses (25 mg/kg) apparently does not affect bone resorption or remodeling, as one would expect if TRAIL protein interacted with OPG in vivo (Walczak et al., 1999). TRAIL exists as a membrane-bound protein on expressing cells in vivo, and OPG interaction with native TRAIL may regulate important cellular functions. Several lines of evidence provide definitive evidence of the role of ODF/OPGL/TRANCE/RANKL as the key factor that regulates osteoclastogenesis (referred herein as RANKL for continuity). First, recombinant RANKL binds specifically and with high affinity to biologically active forms of OPG, but not inactive OPG mutants (Yasuda et al., 1998; Lacey et al., 1998). Second, either native cell surface expressed RANKL, or recombinant soluble RANKL acts as a potent osteoclast differentiation factor in vitro using

either murine or human hematopoietic precursors. Osteoclastogenesis in vitro is dependent on the presence of CSF-1, and is characterized by induction of the expression of key markers that typify the osteoclast cell lineage. This includes the calcitonin receptor, TRAP, cathepsin K, and the integrin v 3, as well as the formation of large multinucleated cells that are capable or resorbing bone. RANKL not only stimulates osteoclast differentiation, but also mature osteoclast activation (Fuller et al., 1998; Burgess et al., 1999) and survival (Jimi et al., 1999) in vitro and in vivo. Third, using fluorescenated RANKL as a probe, all of the hematopoietic progenitors capable of giving rise to osteoclasts during in vitro culture could be isolated and purified from bone marrow cells by flow cytometry (Lacey et al., 1998). Finally, expression of RANKL in osteoblasts was induced by agents that increase osteoclast development in vitro, such as vitamin D3, IL-1 and IL-11, PGE2, and PTH (Yasuda et al., 1998b). Interestingly, some of these agents also appear to simultaneously downregulate or inhibit OPG expression in osteoblasts (Vidal et al., 1998a). This raises the possibility that coordinate regulation of OPG and OPGL expression by osteotropic and calciotropic hormones may be a mechanism used to couple osteoblast and osteoclast development and activation during bone remodeling. Thus, the combination of cell biological data measuring osteoclast differentiation and activation activity, its ability to tightly bind to OPG, and its relatedness to TNF family members all indicated that OPGL is a critical factor that regulates osteoclastogenesis. OPG binds to soluble RANKL in the 30±100 pM range using protein±protein interaction assays in solution, and in the 1 nM range using BIAcore analysis. OPG/TRAIL interactions have been measured in the 1±3 nM range in solution assays.

Cell types and tissues expressing the receptor See Table 1.

Regulation of receptor expression The mouse OPG transcript is expressed in the cartilaginous primordia of bone during embryonic development, then later is expressed in the intestine, kidney, lung, and bone (Simonet et al., 1997). In humans, OPG expression in tissues is detected at relatively high levels in the lung, kidney, intestine, spleen, thymus, and heart (Simonet et al., 1997; Yasuda et al., 1998a). OPG expression can be

1704 William J. Boyle Table 1 Cell types and tissues expressing the receptor Organs and tissues

Cartilaginous bone primordia during fetal development Intestinal epithelium Kidney Liver Lung Thyroid Bone and growth plate cartilage Lymph node Spleen Thymus Heart

Cell types

Hypertrophic chondrocytes Osteoblastic stromal cells Osteoblasts T cells (subset undefined) B cells Intestinal epithelium (small and large) M cells

OPG (Yun et al., 1998). Cytokines such as TNF and TNF , as well as interleukins IL-1, upregulate the production of OPG in an osteosarcoma cell line and in osteoblast-like stromal cells (Vidal et al., 1998b; Brandstrom et al., 1998), indicating a possible link between immune system responses and the regulation of bone metabolism.

Release of soluble receptors OPG is a secreted protein and is not a cell-associated, transmembrane signaling receptor. Analysis of the OPG gene has not revealed any alternatively spliced forms that contain a transmembrane domain or cellanchoring motifs.

SIGNAL TRANSDUCTION

Associated or intrinsic kinases OPG is a cytokine antagonist that can block RANKL binding to RANK, and induction of signal transduction. See the chapter on RANK for discussion on cytoplasmic factors and signaling from this receptor.

Follicular dendritic cells Cell lines

CTLL-2 (IL-2-dependent T cell line) IMR-90 (human diploid lung fibroblasts)

DOWNSTREAM GENE ACTIVATION

LIM 1863 (human colorectal carcinoma)

Transcription factors activated

MG63 (human osteosarcoma)

OPG, RANKL, and RANK have all been implicated as the key extracellular proteins that coordinately interact to regulate osteoclast differentiation and activation. Each of these proteins has been evaluated in mouse molecular genetic models, and their interrelated functions during osteoclastogenesis have been confirmed. A mechanistic model for regulation of osteoclastogenesis can be derived that invokes binding of these proteins in a selective fashion to produce negative or positive regulation of bone mass (Figure 4). RANKL is a pathway agonist that activates the RANK receptor on osteoclast precursors, or on mature osteoclasts, resulting in bone resorption. OPG is a cytokine antagonist that acts as a negative regulator of osteoclast development and activation by sequestering its ligand. The resulting effect in vivo is to block bone resorption, leading to the accumulation of bone mass. Differential expression of OPG and RANKL by the osteoblast is under the control of a diverse collection of hormones and cytokines that influence the rate of bone remodeling,

detected in osteoblastic stromal cells, and is found to be regulated by cytokines, growth factors, and steroid hormones, suggesting a role as a factor that regulates osteoblast and osteoclast coupling during bone development and remodeling (Yasuda et al., 1998b; Vidal et al., 1998b). Members of the TGF superfamily, such as TGF 1 and BMP-2 can induce expression of OPG (Hofbauer et al., 1998, Takai et al., 1998; Horwood et al., 1998). Calciotropic agents known to induce bone resorption, such as vitamin D3, PGE2, and hydrocortisone, can also downregulate OPG expression in osteoblasts (Vidal et al., 1998a). OPG transcripts have also been detected in dendritic cells and osteoblastic sarcomas (Yun et al., 1998; Vidal et al., 1998b). Its expression in some lymphoid cells is upregulated by the ligation of CD40 receptor, a TNFR protein that is closely related to

Osteoprotegerin 1705 Figure 4 Molecular mechanism for the regulation of osteoclast differentiation and activation via OPGL and OPG on osteoclast precursors. OPGL (RANKL) is represented as a transmembrane and soluble protein that binds to and activates the osteoclast receptor RANK, stimulating signal transduction leading to the induction of osteoclast specific gene expression. OPG is a secreted neutralizing or decoy receptor that functions by binding to and sequestering OPGL, thus rendering it unable to activate RANK and thereby preventing the induction of osteoclastogenesis. The membranebound arrowhead-shaped complex represents the putative OPGL convertase activity detected in human 293 fibroblasts that liberates soluble OPGL. TRAF 2, 5, 6 are TNFR-associated cytoplasmic factors involved in signal transduction. TRAP, tartrate-resistant acid phosphatase; CTR, calcitonin receptor; v 3, osteoclast-specific integrin v 3; CatK, osteoclast-specific protease cathepsin K

OPG

and can control the numbers and activity of osteoclasts. At the level of the bone, RANKL would appear to control the coupling of osteoblast and osteoclast functions to coordinate bone remodeling, while OPG can uncouple this process and block bone remodeling. There is still much to earn about this pathway, and exactly how RANKL/RANK interactions can produce changes in gene expression patterns in hematopoietic progenitors to result in the osteoclast cell phenotype. Further study of this pathway should provide insights as to how bone density is regulated by cytokines and hormones during normal development and disease.

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors Our current view of OPG is that it is not activated like traditional receptors that are cell bound. Since OPG is a secreted protein, its activity is regulated at the level of OPG expression and secretion. Overexpression of OPG blocks osteoclast-mediated bone resorption, and leads to increases in bone mass (see below).

Phenotypes of receptor knockouts and receptor overexpression mice The biological activity of OPG was determined in vivo by analyzing the effects of systemic administration of OPG via transgenic delivery of the rat and mouse cDNA into mouse (Simonet et al., 1997). This system employed the use of the ApoE promoter and hepatocyte control element to produce high levels of OPG protein in the liver beginning around day one of postnatal development, and continuing throughout adulthood. Overexpression of OPG in this way is designed to mimic the chronic systemic administration of protein, and therefore would perturb any OPG-regulated process and lead to an identifiable phenotype. Transgenic OPG founder mice expressing varying levels of OPG were found to be healthy, and appeared of normal size and weight. They had no abnormalities in blood cell levels or in serum chemistry. They were able to breed, and readily gave rise to transgenic mouse lines. However, they all had a noticeable phenotype that correlated in severity with the level of exogenous OPG measured in their circulation. OPG transgenics had increased bone density (osteopetrosis), resulting from the accumulation of newly synthesized bone. The bones were of normal length and shape, suggesting that bone growth and modeling was not affected by excess OPG. The increase in bone mass detected in transgenic animal was restricted to the endosteal regions of the trabecular or

1706 William J. Boyle marrow-containing bones. Within the endosteal region, there were few if any TRAP-positive multinucleated osteoclasts observed, suggesting a defect in bone resorption. Comparison of bone sections obtained from OPG transgenic mice and control littermates indeed showed that OPG transgenics lacked mature osteoclasts within the endosteal regions of the affected bones. Normal osteoclast progenitor cells can be detected in the spleens of these mice using in vitro osteoclast formation assays, and immunohistochemistry of the spleen indicated normal expression of the myelomonocytic cell surface marker F4/80 (Austyn and Gordon, 1981). F4/80 is a surface antigen associated with macrophage and osteoclast precursors, and its abundant expression in the spleens of these animals indicates normal development in the myeloid cell lineages. This indicates that the bone phenotype seen in OPG transgenic phenotype is not due to alterations in hematopoiesis leading to the production of the osteoclast progenitor, yet rather these mice harbor a defect in the latter stages of osteoclast maturation. The OPG transgenic mice still produce and harbor osteoclast progenitor cells that could differentiate in vitro after being removed from exogenous OPG exposure in vivo. This means that hematopoiesis leading to the production of osteoclast precursors is unaffected by OPG, and these precursor cells are blocked in their ability to differentiate along the terminal osteoclast maturation pathway (Figure 5). From this work, we now know that OPG impacts a crucial regulatory step during osteoclast development, and that further study of this novel protein will lead to a better understanding of this process, and physiological cues that help to regulate bone density. The murine and human OPG gene has been isolated and partially characterized (Morinaga et al., 1998; Bucay et al., 1998). Using the murine gene, OPG knockout mice have been generated and analyzed to further characterize the function of OPG (Bucay et al., 1998; Mizuno et al., 1998). OPGÿ/ÿ mice were born live, and found primarily to have early onset osteoporosis that increases in severity during aging. These mice have extremely fragile bones, and multiple long bone fractures can be readily observed upon X-ray examination of mice maintained with normal handling. Histological examination of the bones of OPGÿ/ÿ mice shows a dramatic decrease in trabecular and cortical bone mass. The cortical region of the long bones degenerates over time into a trabecularized structure with woven matrix, indicating a very weak form of bone in these animals. Interestingly, the heterozygous OPGÿ/‡ mice also have decreased bone mass, at a level intermediate to that of ‡/‡ and ÿ/ÿ mice (Bucay

Figure 5 OPG effect on osteoclastogenesis in vitro. Diagram depicting the hypothetical action of OPG on osteoclast development in vitro using the bone marrow and stromal cell co-culture system. Bone marrow precursor cells treated with M-CSF (CSF-1) give rise to bone marrow macrophage cells capable of differentiating into mature osteoclasts. Coculture of bone marrow macrophages with ST-2 stromal cells in the presence of 1,25 dihydroxyvitamin D3 and dexamethasone differentiate into mature osteoclasts, Prostaglandin E2 (PGE2) potentiates osteoclast formation in this system. OPG blocks osteoclast development at the stage which requires stromal cells, vitamin D3, and dexamethasone. F4/80, a monocyte/macrophage surface marker; TRAP, tartrate-resistant acid phosphatase; CR, calcitonin receptor; v 3, osteoclast specific integrin v 3; CFU-S; colony-forming unit stem cells; CFUGM, colony-forming unit granulocyte/macrophage.

et al., 1998). If the OPG gene dosing levels seen in OPG transgenic, wild-type, and OPGÿ/‡ and ÿ/ÿ mice are compared, it can readily be concluded that the level of OPG directly determines the level of bone mass, implying that OPG is a secreted molecule that regulates bone density. OPGÿ/ÿ mice have also been shown to have calcifications in the vessel walls of major arteries in the heart and kidney (Bucay et al., 1998). The underlying mechanism of this effect is not known, but may be related to secondary events that occur following losses in bone mineral density. The knockout of the OPG actually provided a phenotype in mice that is diametrically opposite that of OPG

Osteoprotegerin 1707 overexpression, reinforcing the concept that it acts to regulate bone mass as a negative regulator of osteoclast activity.

Human abnormalities To date, there have been no reports of any structural abnormalities in the OPG gene or protein that provide any obvious links to the pathophysiology of human disease.

THERAPEUTIC UTILITY

Effect of treatment with soluble receptor domain The early hypothesis that OPG functions as a secreted osteoclast inhibitor in vivo was tested by administration of recombinant OPG in neonate animals. Young growing mice were injected with recombinant murine OPG protein daily for 3±7 days, and its effects on bone were measured by radiography and quantitative histology (Simonet et al., 1997; Yasuda et al., 1998). In growing mice, newly synthesized bone is actively generated at the growth plates of the long bones, particularly at the proximal tibial metaphysis. Injection of OPG leads to the rapid accumulation of bone at this site, coincident with the decreased numbers of TRAP-positive osteoclasts. Microscopic analysis of this area shows the accumulation of newly synthesized bone-encased cartilage, or osteoid. This is the same histologic picture as that seen in OPG transgenic mice, and indicates that the phenotypic effects seen in transgenics can be recreated in normal mice when given intravenous recombinant protein. The effects of recombinant OPG in young growing mice were similar to those seen in mice treated with bisphosphonates (Sietsema et al., 1989), small molecule antiresorptive therapeutics useful for the treatment of osteoporosis. The effects of OPG and the bisphosphonate pamidronate were tested in this in vivo model, and both compounds were found to lead to equivalent changes in the accumulation of bone mass (Simonet et al., 1997). Thus, the biological activity of OPG on osteoclast maturation acts like an antiresorptive agent in normal animals. OPG can also block bone resorption in a disease model characterized by increased osteoclast numbers and/or activity. A rat model for osteoporosis associated with the loss of estrogen was used to compare the effects of OPG with those of pamidronate. In this model, ovairectomy leads to the rapid loss of bone mass due to increased osteoclast activity (Kalu et al.,

1989). OPG and pamidronate both blocked increases in osteoclast numbers and activity, and prevented bone loss seen in the untreated ovariectomized rat controls. OPG has also been analyzed in the thyroparathyroidectomized rat model (Yamamoto et al., 1998). In this model, serum calcium levels are raised by the exogenous addition of either parathyroid hormone or vitamin D3 via induction of osteoclastmediated bone resorption. OPG can both block the induction of serum hypercalcemia in a rapid manner and reverse it once established, suggesting an immediate effect on osteoclast function and/or survival. These data indicate that the OPG bioactivity translates into a protective agent that may be useful for the treatment of osteopenic disorders characterized by excessive bone loss due to elevated osteoclast numbers or activity, and may have important implications for the clinic. OPG is currently being tested in phase I/II clinical trials in the United States, and single-dose injections have been shown to suppress surrogate bone resorption markers in normal postmenopausal women (Bekker et al., 1999).

Effects of inhibitors (antibodies) to receptors There have been no reports of molecules that inhibit OPG bioactivity. The analysis of OPG knockout mice provides insight into the effects that OPG inhibition would probably produce in vivo (see above). OPG neutralizing antibodies have been isolated, and have been shown to block OPG effects on osteoclast development in vitro (Yasuda et al., 1998a).

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LICENSED PRODUCTS OPG recombinant protein and antibodies for research Alexis Biochemicals, 6181 Cornerstone Court East, Suites 102±104, San Diego, CA 92121, USA R&D Systems, Inc., 614 McKinley Place N.E., Minneapolis, MN 55413, USA Santa Cruz Biotechnology, Inc., 2161 Delaware Avenue, Santa Cruz, CA 95060, USA