MEDSCAPE

The Discovery of New Members of the TNF Receptor and

Ligand Families and Their Relationship to Osteoclasts and
Bone Metabolism
The Discovery and Biologic Activity of Osteoprotegerin (OPG)
In 1997, 3 laboratories, working on different questions and using different starting
points and different approaches, independently identified the same protein that has
important implications for both basic bone biology and therapeutics. They were
followed in 1998 by another lab that identified the same protein from yet another
source. During a sequencing project of rat intestinal cDNA, Simonet and
colleagues[19] identified a new molecule, which by sequence homology, was found to
be a novel member of the TNF receptor (TNFR) superfamily. The biologic activity of
this glycoprotein was tested in transgenic mice overexpressing it in the liver, which
resulted in high levels of the new protein in the blood: The mice developed severe
osteopetrosis of both femurs and vertebrae as a result of markedly reduced
osteoclast number, with no concomitant reduction in macrophage number. When the
soluble factor was added to the co-culture model of hematopoietic cells and stromal
cells, it dose-dependently inhibited osteoclast formation in the presence of all
stimulators tested, including 1,25(OH) D , PTH, PGE , and IL-11. Consistent with
this, subcutaneous administration of the factor to wild-type mice resulted in
increased bone mass. Strikingly, it also protected against the decrease in bone
mass that occurs in the ovariectomized/estrogen-deficient rat model of
postmenopausal osteoporosis. On the basis of its ability to "protect bone," this new
TNFR family member was dubbed "osteoprotegerin" or OPG. [19]
2

At the same time, Tsuda and coworkers[20] used a protein purification approach to
isolate a heparin-binding protein from the conditioned medium of human fibroblasts;
they termed the factor "osteoclastogenesis inhibitory factor" (OCIF) because it
inhibited osteoclast formation. Shortly thereafter, they showed that the cDNA
sequence for OCIF was identical to that of OPG. [21] In the same year, Tan and
colleagues[22] searched a database of expressed sequence tags and identified a new
member of the TNFR family, which they called TR1 (TNF receptor-like molecule 1);
TR1 was identical to OPG in sequence and in those activities tested (inhibition of

osteoclast formation in co-cultures, inhibition of resorption pit formation by

osteoclasts in vitro, and inhibition of bone resorption in organ cultures of fetal mouse
long bones).[23] Finally, Yun and colleagues[24] cloned a molecule from a follicular
dendritic cell (FDC)-like cell line FDC-1, named it FDC receptor-1 (FDCR-1), and
found by sequencing that it was identical to OPG.
Thus, OPG/OCIF/TR1/FDCR-1 are the same molecule and will be referred to
hereafter as OPG for both convenience and because the name embodies the
biologic activity of importance for the discussion that ensues. The use of this term
has been recommended by Suda and colleagues,[4] and a President's Committee
established by the American Society for Bone and Mineral Research
(ASBMR; http://www.asbmr.org) is currently meeting to discuss a common
nomenclature for the field for OPG and other TNFR-ligand family members (see also
below).
Inactivation of OPG by homologous recombination in mice confirms that the function
of this secreted molecule is to block osteoclast formation and bone resorption. OPGdeficient (OPG -/-) mice develop severe early onset osteoporosis, with increased
osteoclast numbers, marked cortical and trabecular bone porosity, decreased
strength and bone density, and increased fracture incidence. [25,26] After 2 months of
age, markedly increased bone turnover is seen, with increased osteoclastic and
osteoblastic activity.[25] Adult OPG-deficient mice display serial vertebral compression
fractures and collapsed femur epiphyses. OPG -/- mice die at approximately 6
months of age from complications associated with the severe skeletal abnormalities
and calcification of the aorta and renal arteries, the latter observation suggesting
that a normal OPG function may be to prevent calcification of larger arteries. [25] This
is of particular interest because the degrees of vascular calcification and
osteoporosis are correlated in aging women.[27]
Although OPG is made by osteoblastic cells, it is not restricted to bone tissue and
indeed is expressed in a wide variety of cell and tissue types, including the aorta and
other large arteries, consistent with the fact that calcification of these latter sites
occurs when OPG is absent. Its multiple other roles and activities are currently the
subject of intense investigation, especially in T and B cells, in which it appears to
play a crucial role in early development and activity of these lymphocytes (reviewed
by Kong and coworkers[8]). It is interesting to note that the mouse, rat, and human
OPG sequences are highly conserved. There are many other interesting structural

features of OPG that may contribute to its biologic activities, including the presence
of "death domains" associated with apoptosis. These features are beyond the scope
of this article, however, and interested readers are referred to 2 recent reviews for
more information.[4,28]
The Discovery and Biologic Activity of ODF/OPGL/RANKL
Just as work in several labs and different model systems came together with the
discovery of OPG, so too is the story of the discovery of its ligand intriguing and
multifaceted (reviewed by Suda and associates [4] and Jurdic[29]).
With OPG being a soluble member of the TNFR family, the search for its ligand was
predicated on the notion that it might logically be a membrane-bound molecule. One
likely ligand source was stromal cells, given OPG's ability to inhibit
osteoclastogenesis in their presence. This thinking induced Yasuda and colleagues
to use expression cloning to search for OPG-binding molecules expressed by a
1,25(OH) D -treated stromal cell line known to support osteoclastogenesis. They
called the molecule they isolated "osteoclast differentiation factor" (ODF) on the
basis of its ability to support osteoclastogenesis in cell cultures as well as in bone
organ cultures, in which calcium-45 release was concomitantly stimulated. [30] As
predicted, expression of ODF by osteoblasts/stromal cells was stimulated by all the
hormones [eg, 1,25(OH) D , PTH, PGE ] known to stimulate osteoclast formation.
2

Expression cloning was also used by Lacey and coworkers [31] to isolate an OPGbinding ligand (OPG-L; OPGL) from a myelomonocytic cell cDNA library. OPGL
displayed the same kinds of activities as described above for ODF; that is, it
stimulated osteoclast development in bone marrow cultures, stimulated formation of
resorption pits by osteoclasts isolated from rat bones, and induced hypercalcemia in
mice. These data suggested that OPGL stimulated both osteoclast differentiation
and osteoclast activity. When OPGL was deleted from mice by homologous
recombination, the OPGL -/- mice displayed classic and severe osteopetrosis, with a
complete lack of osteoclasts, total occlusion of the bone marrow space within
endosteal bone, and a defect in tooth eruption. [32] OPGL -/- mice have normal
osteoclast precursors, suggesting that OPGL is an absolute requirement for
osteoclast development. OPGL -/- mice also exhibit defects in early differentiation of
T and B cells. For many years, potential relations between bone and immune cell

activity have been discussed; the discovery of OPG and OPGL has brought some
molecular understanding to these relations (see below).
The sequencing of both OPGL and ODF revealed that the molecule was identical to
another already and twice independently cloned member of the TNF ligand family,
called TRANCE (TNF-related activation-induced cytokine) or RANKL (receptor
activator of NF-kappaBligand). This identity contributed substantially to knowledge of
potential activities and pathways impinged on by ODF/OPGL. TRANCE, an early
gene that is induced by stimulation of the T-cell receptor, was cloned during a search
for apoptosis-regulatory genes in murine T-cell hybridomas and is known to be
linked to the JNK (c-Jun N-terminal kinase) kinase-apoptosis pathway in T-cells and
dendritic cells.[33] RANKL was discovered in a search for proteins that would bind to
RANK, a TNFR family member that is ubiquitously expressed (but highly expressed
in muscle and thymus) and implicated in differentiation of monocytes and dendritic
cells, consistent with the phenotype found in OPGL -/- mice. RANK, originally
described as a receptor on T cells and dendritic cells, [34] is identical to the receptor on
osteoclasts that binds RANKL and also called "osteoclast differentiation and
activation receptor" (ODAR).[35] As we indicated earlier for OPG, it is confusing and
unwieldy to continue to list multiple acronyms/names for these other important
molecules. For the remainder of this article, we will refer to
ODF/SOFA/OPGL/RANKL/TRANCE as RANKL and to RANK/ODAR as RANK.
Thus, the following paradigm can now be put forward as the molecular
underpinnings of the cell-cell interactions underlying osteoblast/stromal cell
regulation of osteoclastogenesis. RANKL, expressed on the cell surface of
osteoblasts/stromal cells, binds to RANK, expressed on the cell surface of
hemopoietic osteoclast precursor cells, inducing a signaling and gene expression
cascade that leads to their differentiation and maturation to osteoclasts. OPG, which
can bind to RANKL, acts as a decoy receptor, blocking the interaction between
osteoblasts/stromal cells and the osteoclast precursors, thereby inhibiting osteoclast
formation (Table 1 and Figure 2). In the presence of the permissive factor M-CSF,
RANKL is both necessary and sufficient for the differentiation of osteoclast
precursors to mature, activated osteoclasts, without the need for addition of cocultures or other cytokines, hormones, or growth factors. Although RANKL is a
membrane-bound ligand, soluble RANKL can also support osteoclast formation,
consistent with some findings that the conditioned medium of osteoblasts/stromal
cells will support osteoclastogenesis in at least some systems. As already raised,

RANKL has capacity to activate osteoclasts, stimulating inactive osteoclasts to bone

resorption,[31,36,37] and to inhibit osteoclast apoptosis[36] (Figure 1). Treatment of mice
with RANKL results rapidly (1 day) in dose-dependent, severe hypercalcemia and
within 3 days leads to marked bone loss, with more differentiated osteoclasts and
increased bone resorption surface and without an increase in osteoclast number;
these effects can be blocked with OPG. [31]

Figure 2.
Regulation of osteoclastogenesis by factors from osteoblasts/stromal cells interacting with receptors
on hematopoietic osteoclast precursor cells. RANK-L: receptor activator of NF-kappaB ligand;
RANK: receptor activator of NF-kappaB; OPG: osteoprotegerin; M-CSF: macrophage-colony
stimulating factor.

Identification of these new TNF receptor-ligand family members is also likely to shed
new light on other aspects of osteoblast-osteoclast interactions. For example, Cbfa1,
the recently identified transcription factor controlling osteoblast development, [38] may
be one regulator mediating the observed coupling between osteoblastogenesis and
osteoclastogenesis. A Cbfa1 response element has been found in the promoter of
the mouse RANKL gene.[39] Embryonic mice lacking Cbfa1 are also deficient in
RANKL mRNA in developing long bones, and calvarial cells from these mice have
an impaired ability to support osteoclastogenesis. [40]
As with OPG, expression of RANKL mRNA is widely distributed but the highest
levels are found in the skeleton (trabecular bone and bone marrow; in adult bone, in
growth plate and in the metaphyseal periosteum [31]) and in lymphoid tissues (lymph
node, thymus, spleen, fetal liver, and Peyer's patches), consistent with the
osteoclast and T- and B-cell deficiencies found in RANKL -/- mice. However,
whereas the mRNA for RANK is widely distributed, the distribution of RANK protein
appears restricted to osteoclasts and osteoclast lineage cells, dendritic cells, B- and
T-cell lines, and fibroblasts.[35] With RANK and RANKL expression as described, it is
interesting to note that animals injected with RANKL form osteoclasts only in bone
and not in other tissues that also likely contain permissive levels of M-CSF.[31] This
suggests that there may be other factors in the bone microenvironment (and in
vitro culture models) that are necessary for osteoclast differentiation from precursor
cells.
Binding of RANKL to RANK initiates a signaling cascade, which includes activation
of NF-kappaB (the factor whose ablation leads to osteopetrosis) and the JNK kinase
pathway. These pathways and their regulators and mediators are being intensely
studied not only in osteoclast lineage cells but in a variety of other cells that express
RANK. It thus seems reasonable to speculate that future therapies may be based on
modulation of these pathways.
OPG and RANKL Are Regulated by Systemic Hormones, Growth Factors, and
Cytokines
The striking and opposite phenotypes of OPG-overexpressing mice (osteopetrosis;
also seen with RANKL deletion) and OPG-deficient (osteoporosis; also seen with
RANKL overexpression) make OPG and RANKL candidates for being important
mediators for the stimulatory or inhibitory effects of a variety of systemic hormones,

growth factors, and cytokines on osteoclastogenesis. In addition, several of the

same regulators that stimulate the "positive" osteoclast differentiation signal,
RANKL, concomitantly inhibit the "negative" signal, OPG, in osteoblasts/stromal
cells. These regulators include 1,25(OH) D , PTH, PGE , and IL-11, suggesting that
hormonally regulated osteoclastogenesis requires a critical balance between
production of RANKL and its endogenous receptor antagonist, OPG [4,41] (Table 2 and
Figure 2). There is evidence to support the view, recently called "the convergence
hypothesis," that the activity of the proresorptive and antiresorptive agents
"converges" at the level of these 2 mediators, whose final ratio controls the degree
of osteoclast differentiation, activation, and apoptosis. [41]
2

Estrogen. Estrogen withdrawal after ovariectomy or menopause leads to an

increase in growth factors, such as M-CSF, and inflammatory cytokines, such as IL1, IL-6, and TNF, with concomitant increased recruitment of myeloid progenitors,
osteoclast formation, and bone resorption. Estrogen deficiency also increases
activation[42] and reduces apoptosis[43] of mature osteoclasts. Reciprocally, estrogen
replacement reverses these effects and has a bone-protective effect. In recently
published results, Hofbauer and colleagues found that estrogen stimulates
production of OPG in osteoblastic cells[44]; as mentioned earlier, OPG treatment of
oophorectomized rats completely prevents bone loss. [19] Together, these results
suggest that enhancement of OPG may play a major role in the antiresorptive action
of estrogen on bone. In a study presented at a recent meeting of the ASBMR,
estrogen was found to be an inhibitor of RANKL and RANKL-stimulated
osteoclastogenesis.[45] In another paper presented at the ASBMR meeting, estrogen
was found to decrease the responsiveness of osteoclast precursors to RANKL by
downregulating RANKL-induced JNK activity.[46] All of these data are consistent with
multiple roles of estrogen: to regulate resorption-stimulating cytokine expression and
OPG production by osteoblasts/stromal cells and to act directly on osteoclast
progenitors to inhibit RANKL-induced and M-CSF-induced osteoclast differentiation.
Glucocorticoids. Emerging data with glucocorticoids are also of interest. Systemic
glucocorticoid administration results in increased (or sustained) bone resorption and
a decrease in bone formation, leading to osteoporosis. [47,48] Mechanisms underlying
the increased osteoclastogenesis have been unclear, but it is interesting to note that
recent data show that glucocorticoids reduce OPG expression in human osteoblastic
cells in vitro.[49] As important, the decrease in OPG is more marked than the

glucocorticoid-induced increase in RANKL, which leads to an increased

RANKL/OPG ratio; this change in RANKL/OPG ratio may mediate the proresorptive
effect of glucocorticoids.[50] Interestingly, preliminary data suggest that serum OPG
concentrations are reduced significantly in patients undergoing systemic
glucocorticoid therapy.[51]