RANK Ligand

10 Pages·0.137 MB·English

by  Boyle W.J.

RANK Ligand
William J. Boyle
*
Department of Cell Biology, Amgen Inc., One Amgen Center Drive, Thousand Oaks,
CA 91320-1799, USA
*corresponding author tel: [phone], fax: [phone], e-mail: [email protected]
DOI: 10.1006/rwcy.2000.05010.
SUMMARY literature as a T cell protein (TRANCE) whose
expression was controlled by calcineurin-regulated
RANK ligand (RANKL) is a TNF-related protein transcription factors during activation (Wong et al.,
that binds to, and activates, the TNFR-related 1997). Anderson et al. (1997) subsequently reported
protein known as RANK. Several groups studying the molecular cloning of a novel TNF receptor-
both immune homeostasis and bone metabolism related protein, called RANK, and a cognate ligand,
independentlyidentifiedthisligand/receptorpair.The termedRANKL.Yasudaetal.(1998)andLaceyetal.
expression of RANKL suggests a role in the inter- (1998) both reported the identification and cloning
action of T cells and antigen-presenting cells during of a long sought after biological entity known as
generation of immune responses, and in regulating osteoclastdifferentiationfactor.Bothgroupshadused
bone resorption processes within the primary spongi- osteoprotegerin (OPG) as a probe to identify this
osa and cartilaginous growth plate. RANKL/RANK factor, which was termed OPGL or osteoclast differ-
interactions stimulate signal transduction leading to entiation factor (ODF). TRANCE, RANKL, ODF,
activationoftheNF(cid:20)BandJunkinasepathways,and and OPGL are all identical to each other, and the
initiategeneexpressionpatternsthattypifytheosteo- methods used to identify each has helped shape our
clast lineage. understanding of the interesting biology that sur-
Overexpression of RANKL in mice is associated rounds this molecule.
with increased bone resorption and loss of bone
density,whilelossofRANKLresultsinanabsenceof
Alternative names
the osteoclast lineage and an absolute defect in bone
resorption.TheexpressionofRANKLisregulatedby
calciotropic hormones and proresorptive cytokines RANK ligand is also known as TRANCE (TNF-
that stimulate bone resorption during physiologic related activation-induced cytokine) (Wong et al.,
and pathophysiologic conditions. Thus, RANKL 1997),ODF(osteoclastdifferentiationfactor)(Yasuda
ligand is essential for regulating bone density via the et al., 1998), and OPGL (osteoprotegerin ligand)
osteoclast and is implicated in osteopenic disorders (Lacey et al., 1998).
where increased osteoclast activity is observed, such
as osteoporosis and rheumatoid arthritis.
Structure
The human RANKL is a type II transmembrane
BACKGROUND
protein of 317 amino acid residues. The N-terminal
region of the protein encodes a short intracellular
Discovery
domain, followed by a hydrophobic transmembrane
domain. The C-terminal region encodes the extra-
The tumor necrosis factor (TNF)-related protein cellular domainofRANKL.Thisregioniscomposed
known as RANK ligand (RANKL) was identified by ofastalkofabout70aminoacids,followedbyaTNF
four independent groups. It was first reported in the core region of about 158 amino acids. The TNF core
490 William J. Boyle
is composed of 10 (cid:12) sheet-forming sequences with Chromosome location
short loop regions that separate the (cid:12) sheets. The
TNF core has homology with all members of the
The human RANKL gene is localized on chromo-
TNF family and contains motifs characteristic of
some13q14(Andersonetal.,1997;Laceyetal.,1998).
the family (Smith et al., 1994)
Main activities and Relevant linkages
pathophysiological roles
The are no other known TNF-related proteins that
clustertothisregionofchromosome13,andthereare
The name RANKL stems from the molecule’s ability
no known genes involved in bone metabolism or
to act as a ligand for the TNFR-related protein
immune homeostasis that are localized to this same
RANK. RANK-expressing cells treated with soluble
region.
RANKL stimulate signal transduction leading to the
activation of NF(cid:20)B (Anderson et al., 1997; Darney
et al., 1999; Hsu et al., 1999). Since RANK was first Regulatory sites and corresponding
identified as a dendritic surface receptor, a potential
transcription factors
role in modulating dendritic cell differentiation and
survival was tested. Soluble RANKL can act as a
costimulatory factor during antigen presentation in The transcription factors that mediate the induction
in vitro culture (Anderson et al., 1997), and impacts of RANKL mRNA expression in various cell types
the survival of dendritic cells during in vitro culture havenotbeenidentified.Gaoetal.(1998)havefound
(Wong et al., 1997). These studies indicated a poten- that the osteoblast-specific transcriptional regulator
tial role for RANKL during the regulation of adap- Cbfa1, an essential factor controlling osteoblast dif-
tive immune responses and in immune homeostasis. ferentiation, controls expression of RANKL mRNA.
RANKLis the critical factorcontrolling osteoclast The promoter region of this gene is believed to
differentiation and activation (Lacey et al., 1998; contain consensus-binding sites for this factor, but
Yasudaetal.,1998;Kongetal.,1999a).Furthermore, there have been no published reports characterizing
the TNFR-related protein RANK has also been these sites to date.
identified as the intrinsic hematopoietic cell surface
determinant that mediates the effects of RANKL,
Cells and tissues that express
and controls bone mass and calcium metabolism
(Nakagawa et al., 1998; Hsu et al., 1999; Li et al., the gene
1999). Addition of soluble RANKL to bone marrow
precursor cells in vitro in the presence of CSF-1,
See Table 1.
stimulates osteoclastogenesis and the activation of
matureosteoclaststoresorb(Hsuetal.,1999;Burgess
et al., 1999). These effects can be antagonized by the
addition of osteoprotegerin, a naturally occurring PROTEIN
secreted RANKL antagonist (Simonet et al., 1997).
RecombinantsolubleRANKinducesboneresorption Accession numbers
when administered to rodents, resulting in rapid
increases in serum calcium levels (Lacey et al., 1998).
Human RANKL protein: AAC39731
GENE AND GENE REGULATION
Sequence
Accession numbers
See Figure 2.
Human RANK cDNA: AF053712
Description of protein
Sequence
Like all members of the TNF superfamily, with the
See Figure 1. exception of lymphotoxin (cid:11), RANKL is a type II
RANK Ligand 491
Figure 1 Nucleotide sequence for human RANK ligand.
1 AAGCTTGGTA CCGAGCTCGG ATCCACTACT CGACCCACGC GTCCGCGCGC CCCAGGAGCC
61 AAAGCCGGGC TCCAAGTCGG CGCCCCACGT CGAGGCTCCG CCGCAGCCTC CGGAGTTGGC
121 CGCAGACAAG AAGGGGAGGG AGCGGGAGAG GGAGGAGAGC TCCGAAGCGA GAGGGCCGAG
181 CGCCATGCGC CGCGCCAGCA GAGACTACAC CAAGTACCTG CGTGGCTCGG AGGAGATGGG
241 CGGCGGCCCC GGAGCCCCGC ACGAGGGCCC CCTGCACGCC CCGCCGCCGC CTGCGCCGCA
301 CCAGCCCCCC GCCGCCTCCC GCTCCATGTT CGTGGCCCTC CTGGGGCTGG GGCTGGGCCA
361 GGTTGTCTGC AGCGTCGCCC TGTTCTTCTA TTTCAGAGCG CAGATGGATC CTAATAGAAT
421 ATCAGAAGAT GGCACTCACT GCATTTATAG AATTTTGAGA CTCCATGAAA ATGCAGATTT
481 TCAAGACACA ACTCTGGAGA GTCAAGATAC AAAATTAATA CCTGATTCAT GTAGGAGAAT
541 TAAACAGGCC TTTCAAGGAG CTGTGCAAAA GGAATTACAA CATATCGTTG GATCACAGCA
601 CATCAGAGCA GAGAAAGCGA TGGTGGATGG CTCATGGTTA GATCTGGCCA AGAGGAGCAA
661 GCTTGAAGCT CAGCCTTTTG CTCATCTCAC TATTAATGCC ACCGACATCC CATCTGGTTC
721 CCATAAAGTG AGTCTGTCCT CTTGGTACCA TGATCGGGGT TGGGCCAAGA TCTCCAACAT
781 GACTTTTAGC AATGGAAAAC TAATAGTTAA TCAGGATGGC TTTTATTACC TGTATGCCAA
841 CATTTGCTTT CGACATCATG AAACTTCAGG AGACCTAGCT ACAGAGTATC TTCAACTAAT
901 GGTGTACGTC ACTAAAACCA GCATCAAAAT CCCAAGTTCT CATACCCTGA TGAAAGGAGG
961 AAGCACCAAG TATTGGTCAG GGAATTCTGA ATTCCATTTT TATTCCATAA ACGTTGGTGG
1021 ATTTTTTAAG TTACGGTCTG GAGAGGAAAT CAGCATCGAG GTCTCCAACC CCTCCTTACT
1081 GGATCCGGAT CAGGATGCAA CATACTTTGG GGCTTTTAAA GTTCGAGATA TAGATTGAGC
1141 CCCAGTTTTT GGAGTGTTAT GTATTTCCTG GATGTTTGGA AACATTTTTT AAAACAAGCC
1201 AAGAAAGATG TATATAGGTG TGTGAGACTA CTAAGAGGCA TGGCCCCAAC GGTACACGAC
1261 TCAGTATCCA TGCTCTTGAC CTTGTAGAGA ACACGCGTAT TTACAGCCAG TGGGAGATGT
1321 TAGACTCATG GTGTGTTACA CAATGGTTTT TAAATTTTGT AATGAATTCC TAGAATTAAA
1381 CCAGATTGGA GCAATTACGG GTTGACCTTA TGAGAAACTG CATGTGGGCT ATGGGAGGGG
1441 TTGGTCCCTG GTCATGTGCC CCTTCGCAGC TGAAGTGGAG AGGGTGTCAT CTAGCGCAAT
1501 TGAAGGATCA TCTGAAGGGG CAAATTCTTT TGAATTGTTA CATCATGCTG GAACCTGCAA
1561 AAAATACTTT TTCTAATGAG GAGAGAAAAT ATATGTATTT TTATATAATA TCTAAAGTTA
1621 TATTTCAGAT GTAATGTTTT CTTTGCAAAG TATTGTAAAT TATATTTGTG CTATAGTATT
1681 TGATTCAAAA TATTTAAAAA TGTCTTGCTG TTGACATATT TAATGTTTTA AATGTACAGA
1741 CATATTTAAC TGGTGCACTT TGTAAATTCC CTGGGGAAAA CTTGCAGCTA AGGAGGGGAA
1801 AAAAATGTTG TTTCCTAATA TCAAATGCAG TATATTTCTT CGTTCTTTTT AAGTTAATAG
1861 ATTTTTTCAG ACTTGTCAAG CCTGTGCAAA AAAATTAAAA TGGATGCCTT GAATAATAAG
1921 CAGGATGTTG GCCACCAGGT GCCTTTCAAA TTTAGAAACT AATTGACTTT AGAAAGCTGA
1981 CATTGCCAAA AAGGATACAT AATGGGCCAC TGAAATCTGT CAAGAGTAGT TATATAATTG
2041 TTGAACAGGT GTTTTTCCAC AAGTGCCGCA AATTGTACCT TTTTTTTTTT TTCAAAATAG
2101 AAAAGTTATT AGTGGTTTAT CAGCAAAAAA GTCCAATTTT AATTTAGTAA ATGTTATCTT
2161 ATACTGTACA ATAAAAACAT TGCCTTTGAA TGTTAATTTT TTGGTACAAA AATAAATTTA
2221 TATGAAAAAA AAAAAAAAAG GGCGGCCGCT CTAGAGGGCC CTATTCTATA G
Figure 2 Amino acid sequence for human RANK ligand.
1 MRRASRDYTK YLRGSEEMGG GPGAPHEGPL HAPPPPAPHQ PPAASRSMFV ALLGLGLGQV
61 VCSVALFFYF RAQMDPNRIS EDGTHCIYRI LRLHENADFQ DTTLESQDTK LIPDSCRRIK
121 QAFQGAVQKE LQHIVGSQHI RAEKAMVDGS WLDLAKRSKL EAQPFAHLTI NATDIPSGSH
181 KVSLSSWYHD RGWAKISNMT FSNGKLIVNQ DGFYYLYANI CFRHHETSGD LATEYLQLMV
241 YVTKTSIKIP SSHTLMKGGS TKYWSGNSEF HFYSINVGGF FKLRSGEEIS IEVSNPSLLD
301 PDQDATYFGA
transmembrane protein that is displayed on the sur- followed by a 20 amino acid hydrophobic transmem-
face with the C-terminus facing outwards (Figure 3). brane domain, which functions in localizing the
The N-terminal 50 amino acids of human RANKL mature protein to the cell surface. The C-terminal
form the intracellular domain. It is not known if this portionextendingfromphenylalanine69toaspartate
region of the protein is involved in cell signaling, 317 forms the extracellular domain, which is divided
although several PXXP sequences characteristic of intotworegions;astalkregionfromphenylalanine69
SH3-bindingmotifshavebeenidentified(Laceyetal., toserine158andtheactiveTNF-relatedmoietyfrom
1998). This region of the RANK polypeptide is lysine 159 to aspartate 317. The TNF core region of
492 William J. Boyle
Figure3 Structuralandfunctionaldomains Figure4 Synthesisandposttranslationalcleavageof
ofthehumanRANKL.Theprimaryhuman the RANKL. Illustration depicting the regulation of
RANKL structure. SP, signal peptide; TM, RANKLbiosynthesisinosteoblastsviastimulationof
transmembrane region; TNF core, core the transcriptional factor cbfa1. RANKL mature
region of homology to all TNF family polypeptide is a type II transmembrane protein.
members and active cytokine moiety. Des- RANKLcanbecleavedandreleasedfromexpressing
cendingarrowheadsindicatetherelativesite cells by a TNF-convertase-like activity (Lum et al.,
of proteolytic cleavage. 1999).
the protein is predicted to contain all 10 (cid:12) sheet-
forming sequences present in all known TNF-related
proteins (Banner et al., 1993).
Discussion of crystal structure
No crystal structure has been reported for RANKL.
Important homologies
The mouse and human RANKL cDNAs have been activated T cells and in T cell leukemia (Kong et al.,
cloned and sequenced, and their protein products 1999a). The shed protein is biologically active, as are
compared (Anderson et al., 1997; Lacey et al., 1998). recombinant proteins of similar length made in
The human and mouse proteins are about 85% bacteria or mammalian cells (Lacey et al., 1998).
identical without appreciable gaps in the alignment.
RANKL is found to be most closely related to the
CELLULAR SOURCES AND
apoptosis-inducing cytokine TRAIL (34% similar-
ity), and both proteins are capable of binding to TISSUE EXPRESSION
osteoprotegerin (Emery et al., 1998).
Cellular sources that produce
Posttranslational modifications
See Table 1.
The mouse and human RANKL polypeptide is
modified by N-linked glycosylation (Lacey et al., Eliciting and inhibitory stimuli,
1998). The primary sequence indicates that both the including exogenous and
mouse and human proteins have two acceptor sites,
endogenous modulators
one of which is not conserved in exact location
relative to each other. The N-linked glycosylation
occursatsiteswithintheTNFcoreregion.Themouse RANKligand expression is inducedin osteoblastsby
and human proteins can also be cleaved from the various calciotropic hormones and pro-resorptive
cellsurfacewithinthestalkregion(phenylalanine139 cytokines that induce bone resorption and calcium
for mouse and isoleucine 140 for human) when metabolism(seeSudaetal.,1999forareview).These
expressed in human 293 fibroblasts (Lacey et al., include parathyroid hormone (PTH), parathyroid
1998) (Figure 4). hormone-related peptide (PTHrP), vitamin D3,
TheTNFconvertaseTACEhasbeenimplicatedas glucocorticosteroids, prostaglandin E (PGE ), IL-1,
2 2
a cell-associated protease capable of cleaving mem- and IL-11, and TNF(cid:11). In contrast, estrogen and
brane-boundRANKL(Lumetal.,1999).RANKLis TGF(cid:12) downregulate RANKL expression in osteo-
found to be rapidly cleaved off the surface of blasts, and coordinately upregulate the expression of
RANK Ligand 493
Table 1 Cells and tissues that produce RANK ligand
Tissues Bone marrow
Lymph node
Heart
Intestine
Cells Osteoblasts
Activated T cells
Activated B cells and myeloma cells
Bone marrow precursors (myeloid)
Stromal fibroblasts and synovial cells
Cell lines ST2 (murine osteoblastic stromal cells)
32D (murine myeloid leukemia)
EL4 (murine T cell lymphoma)
7B9 (murine T helper cell line)
70Z/3 (murine pre-B cell line)
KG-1 (human myeloblastic leukemia)
LIM 1863 (human colon carcinoma cell line)
osteoprotegerin (Hofbauer et al., 1998). In T cells, (cid:15) Inductionof matureosteoclast survival(Jimiet al.,
RANKL expression and release from the cell 1999).
membrane is induced during cell activation by liga- (cid:15) Activation of osteoclast-mediated bone resorption
tion of CD3 (Wong et al., 1997; Kong et al., 1999b). (Fuller et al., 1998; Burgess et al., 1999).
TheonlyknownmodulatorofRANKLbioactivity (cid:15) Stimulation of osteoclastogenesis from the murine
is osteoprotegerin, a soluble TNFR-related protein macrophagecelllineRAW264.7(Hsuetal.,1999).
and cytokine antagonist. However, the putative
RANKL convertase (Lacey et al., 1998; Lum et al.,
Regulatory molecules: Inhibitors
1999) may also have a regulatory function by both
and enhancers
releasing RANKL into the circulation and/or down-
regulating surface presentation on expressing cells.
Osteoprotegerin is known to negatively regulate
RANKL bioactivity. No other inhibitors or enhan-
RECEPTOR UTILIZATION
cers have been characterized to date.
RANKL is known to exert its biological effects via
Bioassays used
signaling through the TNFR-related protein RANK.
(cid:15) Vitamin D3-dependent, bone marrow stromal cell
IN VITRO ACTIVITIES co-culturesystemtogenerateosteoclasts(Udagawa
et al., 1989; Lacey et al., 1995).
In vitro findings (cid:15) Bone marrow progenitors cultured in the presence
of CSF-1 and soluble RANKL to generate osteo-
clastsintheabsenceofvitaminD3andosteoblastic
The following is a list of in vitro biological activ-
stromal cells (Lacey et al., 1998; Yasuda et al.,
ities that have been reported as a direct or indirect
1998).
consequence of activating RANK on receptor-
(cid:15) Stimulation of alloreactive T cell proliferation
bearing cells following stimulation with RANKL:
using dendritic cells (Anderson et al., 1997; Wong
(cid:15) Stimulation of alloreactive T cell proliferation et al., 1997).
(Anderson et al., 1997; Wong et al., 1997). (cid:15) Osteoclast colony-forming assay in semi-solid
(cid:15) Activationofosteoclastdifferentiation(Laceyetal., media (Lacey et al., 1998).
1998; Yasuda et al., 1998; Hsu et al., 1999). (cid:15) Osteoclast pit-forming assay on dentine bone slices
(cid:15) Stimulationofosteoclast-likecellcolonyformation (Laceyetal.,1998;Fulleretal.,1998;Burgessetal.,
in semi-solid medium (Lacey et al., 1998). 1999).
(cid:15) Induction of osteoclast-specific gene expression (cid:15) RAW 264.7 cell line assay for in vitro osteoclasto-
(Lacey et al., 1998; Hsu et al., 1999). genesis (Hsu et al., 1999).
494 William J. Boyle
IN VIVO BIOLOGICAL of T cell and B cell development. Dendritic cells
appear normal in these mice, and circulating levels
ACTIVITIES OF LIGANDS IN
of B and T cells are observed in the circulation.
ANIMAL MODELS
RANKL can act as a dendritic cell survival factor
in vitro, an activity that can be complimented by
Normal physiological roles CD40L (Anderson et al., 1997). These data provide a
biological link between the role of RANKL in both
RANKL is required for the differentiation and acti- bone and immune homeostasis, and raises the
vationofosteoclastsandforthedevelopmentoflymph intriguing possibility of a functional link between
nodes (Kong et al., 1999a). In addition, RANKL these two critical organ systems.
plays a role in stimulating of myeloid-derived
dendritic cells to enhance allostimulatory activation
of T cells (Anderson et al., 1997; Wong et al., 1997). Transgenic overexpression
Although RANK was identified as a dendritic cell
receptor, neither RANK nor RANKL is required for
No transgenic animals have been reported in the
normal dendritic cell development and mature func-
literature.
tions. RANKL is also required during T cell devel-
opment (Kong et al., 1999a), although this effect is
not mediated via interactions with RANK (Dougall
et al., 1999; Li et al., 1999). Pharmacological effects
RANKL stimulates the rapid induction of bone
Species differences resorptionand elevates serum calcium when adminis-
tereddailytomiceandratsinthedoserangeof0.05–
Both human and mouse RANKL work equally well 1.0mg/kg (Lacey et al., 1998).
in murine cell-based bioactivities. Conversely, both
murine and human osteoprotegerin bind equally well
to mouse and human RANKL. Murine RANKL is a Interactions with cytokine network
potent stimulator of osteoclastogenesis from human
hematopoietic precursor cells.
Most, if not all calciotropic hormones and pro-
resorptive cytokines have recently been shown to
upregulatemRNAexpressionofRANKLincelllines
Knockout mouse phenotypes
and primary cell cultures (Hofbauer et al., 2000).
Osteoprotegerin, which blocks osteoclastogenesis
Clear evidence as to the role of this molecule in vivo induced by RANKL in rodents, can also inhibit
has been derived from the analysis of mice lacking osteoclast formation and bone resorption induced by
RANKL (Kong et al., 1999a). RANKL(cid:255)/(cid:255) mice treatment with calciotropic factors (Morony et al.,
were born with severe osteopetrosis that did not 1999), suggesting that the RANK signaling pathway
resolve with age, and had other characteristic is the ultimate common mediator of humoral signals
hallmarks seen in other rodent osteopetrosis models, that regulate bone resorption and calcium metab-
such as lack of tooth eruption, club-shaped bones, olism. RANK-knockout mice have been challenged
and splenomegaly. At the histological level, increases with TNF(cid:11), IL-1(cid:12), 1(cid:11),25-(OH) D3, the major
2
in bone mass were due to accumulation of newly calciotropic factors that are known to induce
synthesizedbonesuggestingadefectinresorptionand increasesinboneresorptionandserumhypercalcemia
remodeling. These mice completely lack osteoclasts, (Li et al., 1999). The absence of RANK in these
although normal osteoclast progenitors are present knockout mice prevents the hypercalcemic response
in spleen as detected by in vitro culture assays in the normallyinducedbythesefactors.Interestingly,both
presence of CSF-1 and soluble RANKL. These mice TNF(cid:11) and IL-1(cid:12) administration leads to transient
were found to have an intrinsic defect in the ability hypocalcemia in RANK(cid:255)/(cid:255)mice, suggesting that
of stromal cells to promote osteoclastogenesis via they modulate other mechanisms of calcium homeo-
RANKL, suggesting that it is the sole factor that stasis such as calcium absorption and/or excretion
initiates the osteoclast differentiation program. thatarenormallymaskedbyeffectsofthesecytokines
RANKL(cid:255)/(cid:255)micealsolacklymphnodes,andhave on bone resorption. Surprisingly, challenge of these
hematopoietic cell intrinsic defects in the early stages mice with TNF(cid:11) (1.0mg/kg body weight/day) leads
RANK Ligand 495
to the rare occurrence of osteoclast formation near IN THERAPY
the site of injection, although no significant radio-
graphic or histologic signs of bone resorption are Preclinical – How does it affect
detected. This suggests that TNF can trigger an
disease models in animals?
alternativepathwayleadingtoosteoclastformationin
the RANK knockout mice, presumably by activation
of either TNFR1 and/or TNFR2. RANKL induces potent bone resorption and
increases in blood ionized calcium levels at moderate
doses (Lacey et al., 1998). For this reason it has not
been tested in disease models. In contrast, current
PATHOPHYSIOLOGICAL ROLES preclinical studies are aimed at neutralizing RANKL
bioactivitytoblockpathologicalincreasesinboneloss
IN NORMAL HUMANS AND
due to increased osteoclast activity. See the osteopro-
DISEASE STATES AND
tegerin chapter for a description of the evaluation of
DIAGNOSTIC UTILITY osteoprotegerin in preclinical disease models.
Normal levels and effects Effects of therapy: Cytokine,
antibody to cytokine inhibitors, etc.
RANKL can exist as a cell surface protein and as a
soluble form released by proteolysis, which is pro-
The effects of RANKL antagonists are discussed
bably present in the serum at low amounts under
in the RANK and osteoprotegerin chapters. Yasuda
normal conditions. There have been no reports of
et al. (1998) have shown that polyclonal antibodies
assaysusefulforthedetectionofRANKLbioactivity,
made to soluble RANKL inhibit osteoclastogenesis.
and therefore no correlation has yet been made with
circulating levels of RANK and physiological and/or
pathophysiological states. Pharmacokinetics
There have been no published reports describing the
circulating half-life and bioavailability of recombi-
Role in experiments of nature and
nantsolubleRANKproteinadministeredtoanimals.
disease states
Toxicity
Therehave beennoreportsidentifying animalstrains
as having naturally occurring disruptions in any
aspect of the OPG/RANKL/RANK signaling axis. Recombinant soluble RANKL is a potent stimulator
All three of these genes have been disrupted by of bone resorption. Daily dosing of soluble RANKL
homologous recombination, and their phenotypes at doses in the range of 1.0mg/kg produces severe
have been described in detail (Bucay et al., 1998; hypercalcemia, and is lethal if treatment is extended
Kong et al., 1999a; Dougall et al., 1999; Li et al., for a period of a few days (Lacey et al., 1998).
1999).
Clinical results
Link to Bioassays
There is no current clinical development of this
No assays for human disease states have been protein. Therapeutic drug development is focused on
reported. The following assays would be useful to inhibiting RANKL bioactivity to prevent bone loss.
characterize the role of RANKL in human disease:
(cid:15) ELISA assay to determine circulating levels of
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