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Minireview: The OPG/RANKL/RANK System
SUNDEEP KHOSLA
Endocrine Research Unit, Division of Endocrinology, Metabolism, and Nutrition, Mayo Clinic and Foundation,
Rochester, Minnesota 55905
The identification of the OPG/RANKL/RANK system as the
dominant, final mediator of osteoclastogenesis represents a
major advance in bone biology. It ended a long-standing
search for the specific factor produced by preosteoblastic/
stromal cells that was both necessary and sufficient for oste-
oclast development. The initial cloning and characterization
of OPG as a soluble, decoy receptor belonging to the TNF
receptor superfamily was the first step that eventually led to
an unraveling of this system. Soon thereafter, the molecule
blocked by OPG, initially called OPG-ligand/osteoclast differ-
entiating factor (ODF) and subsequently RANKL, was identi-
fied as the key mediator of osteoclastogenesis in both a mem-
brane-bound form expressed on preosteoblastic/stromal cells
T HE PAST 15 yr have witnessed an explosion in the field
of bone biology. Indeed, the scientific view of bone has
evolved from considering it as a relatively uninteresting scaf-
fold for the rest of the body to an extremely complex organ
regulated by a host of systemic and local factors. The land-
scape of bone biology over this time period has also been
marked by a number of key discoveries that led to opening
up entirely new areas for investigation. These include
(among others) the identification of sex steroid receptors in
bone cells (1, 2); the cloning and characterization of PTH-
related peptide (PTHrP) as the major factor responsible for
the syndrome of humoral hypercalcemia of malignancy (3, 4)
and as an important locally active cytokine in bone, cartilage,
and other tissues (5); the identification of core binding factor
␣1 (Cbfa1) as the (or at least a) master gene controlling
osteoblast differentiation (6, 7); and the important role of
apoptosis in regulating osteoblast and osteoclast number (8).
Certainly, the identification and characterization of the
OPG/RANK-L/RANK system, which began with a seminal
paper published in 1997 (9), is a major addition to this list of
momentous events in bone biology.
OPG: The Key to the Puzzle
OPG was discovered independently by two groups, al-
though the manner in which these groups identified it dif-
fered markedly. The Amgen, Inc. group in the US initially
described a fascinating molecule that they had happened
upon as part of a project characterizing cDNAs in rat intes-
tine (9). Indeed, it is unlikely that this group was actually
searching for OPG, and the project apparently focused on
Abbreviations: Cbfa1, Core binding factor ␣1; JNK, c-Jun N-terminal
kinase; M-CSF, macrophage-colony stimulating factor; NF, nuclear fac-
tor; OCIF, osteoclastogenesis inhibitory factor; OPG-L, ligand for OPG;
PTHrP, PTH-related peptide; TRAF, TNF receptor-associated family;
TRANCE, TNF-related activation-induced cytokine.
5050
Endocrinology 142(12):5050 –5055
Copyright © 2001 by The Endocrine Society
as well as a soluble form. RANKL, in turn, was shown to bind
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its receptor, RANK, on osteoclast lineage cells. The decisive
role played by these factors in regulating bone metabolism
was demonstrated by the findings of extremes of skeletal phe-
notypes (osteoporosis vs. osteopetrosis) in mice with altered
expression of these molecules. Over the past several years,
work has focused on identifying the factors regulating this
system, the signaling mechanisms involved in the RANKL/
RANK pathway, and finally, potential alterations in this sys-
tem in metabolic bone disorders, from the extremely common
(i.e. postmenopausal osteoporosis) to the rare (i.e. familial
expansile osteolysis). (Endocrinology 142: 5050 –5055, 2001)
identifying TNF receptor-related molecules that may poten-
tially have therapeutic utility. One particular cDNA encoded
a truncated TNF receptor-like protein that contained no hy-
drophobic transmembrane-spanning sequences. This clone
would have been no different from other interesting cDNAs
were it not for the fact that transgenic mice overexpressing
this gene had a remarkable skeletal phenotype—they had
marked osteopetrosis. Further analysis of these mice re-
vealed that the osteopetrosis was due to a profound decrease
in osteoclasts, indicating that OPG (short for osteoprotegerin,
i.e. to protect bone) clearly must play a key role in regulating
osteoclastogenesis.
Independently, the Snow Brand Milk Group in Japan re-
ported the identification of the identical molecule (10), but
the route by which they got there was clearly different. In
1981, Rodan and Martin (11) had proposed a novel hypoth-
esis wherein the osteoblast played a central role in mediating
the hormonal control of osteoclastogenesis and bone resorp-
tion. Subsequently, there was accumulating experimental
evidence that osteoblastic/stromal cells were essential for in
vitro osteoclastogenesis and that these cells regulated oste-
oclast differentiation both by producing soluble factors and
also by signaling to osteoclast progenitors through cell-to-
cell contact (12, 13). Thus, the Snow Brand group was sys-
tematically searching for both osteoclast stimulatory and
inhibitory factors. Indeed, they had previously reported the
purification of an osteoclastogenesis inhibitory factor (OCIF)
from the conditioned medium of human embryonic fibro-
blasts (14). They subsequently used the partial protein se-
quence to isolate cDNA clones encoding OCIF, which turned
out to be identical with the protein that had been reported by
the Amgen, Inc. group (9).
These initial reports were followed by numerous studies
further characterizing OPG. It was found to be initially syn-
thesized as a 401 amino acid peptide, with a 21-amino acid
Khosla • Minireview
propeptide that was cleaved, resulting in a mature protein of
380 amino acids (9, 10). As noted earlier, in contrast to all
other TNF receptor superfamily members, OPG lacked trans-
membrane and cytoplasmic domains and was secreted as a
soluble protein. The N-terminal region contained four
cysteine-rich domains (D1–D4) and was most closely related
to TNF receptor-2 and CD40. The C-terminal region con-
tained two death domain homologous regions (D5 and D6)
as well as a region (D7) containing a heparin binding site and
a cysteine residue necessary for homodimerization (9, 10, 15).
OPG mRNA was found to be expressed in a number of
tissues, including lung, heart, kidney, liver, stomach, intes-
tine, brain and spinal cord, thyroid gland, and bone (9, 10).
Because the major biologic action of OPG described to date
has been to inhibit osteoclast differentiation and activity (9,
10), the potential role of OPG in these other tissues remains
to be established. However, mice with targeted ablation of
OPG not only develop severe osteoporosis due to markedly
increased osteoclast formation and subsequent bone resorp-
tion (16, 17), but also have profound calcification of the large
arteries, marked intimal and medial proliferation, and partial
aortic dissection by the age of 4 months (16). Thus, OPG likely
also plays a significant role in the vasculature, and indeed,
a recent study has found that OPG may be an important
survival factor for endothelial cells (18).
Identification of the Ligand for OPG
(OPG-L), RANKL
As soon as OPG was characterized, it became clear that it
likely was going to be the key to identifying the long-sought
osteoclast differentiation factor expressed on osteoblastic/
stromal cells that was essential for osteoclast development.
Indeed, soon after the identification of OPG, both groups had
used expression cloning using OPG as a probe and had
identified its ligand (initially termed OPG-L/ODF) (19, 20),
which turned out to be identical with two previously known
members of the TNF ligand family—TNF-related activation-
induced cytokine (TRANCE) (a gene induced by activation
of the T cell receptor) and RANKL, a factor known to stim-
ulate dendritic cells (21, 22).
Human RANKL is a 317-amino acid peptide that has ap-
proximately 30% homology to the TNF-related apoptosis-
inducing ligand and to CD40, and approximately 20% ho-
mology to Fas ligand (19 –22). It has now been shown to exist
in two forms: a 40- to 45-kDa cellular, membrane-bound form
and a 31-kDa soluble form derived by cleavage of the full-
length form at position 140 or 145 (19). RANKL mRNA is
expressed at highest levels in bone and bone marrow, as well
as in lymphoid tissues (lymph node, thymus, spleen, fetal
liver, and Peyer’s patches) (19 –22). Its major role in bone is
the stimulation of osteoclast differentiation (18, 19), activity
(19), and inhibition of osteoclast apoptosis (23). Indeed, in the
presence of low levels of macrophage-colony stimulating
factor (M-CSF), RANKL appears to be both necessary and
sufficient for the complete differentiation of osteoclast pre-
cursor cells into mature osteoclasts (19, 20). In addition, it is
clear that RANKL (or, as it was originally identified,
TRANCE) has a number of effects on immune cells, including
activation of c-Jun N-terminal kinase (JNK) in T cells (21),
Endocrinology, December 2001, 142(12):5050 –5055 5051
inhibition of apoptosis of dendritic cells (24), induction of
cluster formation by dendritic cells, and effects on cytokine-
activated T cell proliferation (22).
Consistent with these findings, RANKL knockout mice
have severe osteopetrosis with defects in tooth eruption (25).
They also have a complete absence of osteoclasts. In addition,
they exhibit defects in early differentiation of T and B cells,
lack lymph nodes, have defects in thymic differentiation, but
have a normal splenic structure and Peyer’s patches (25). A
somewhat unexpected finding in these mice is that they also
have defects in mammary gland development (26). In par-
ticular, they fail to form lobulo-alveolar structures during
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pregnancy, resulting in death of the newborns (26).
RANK—The Final Piece of the Puzzle
With the identification of the ligand for OPG as being
identical with TRANCE/RANKL, the final piece of the puz-
zle fell into place relatively easily, because the receptor for
this had already been identified as RANK (22). Hsu et al. (27)
subsequently demonstrated that RANK mRNA was highly
expressed by isolated bone marrow-derived osteoclast pro-
genitors and by mature osteoclasts in vivo and that transgenic
mice expressing a soluble RANK-Fc fusion protein had se-
vere osteopetrosis and a skeletal phenotype similar to OPG
transgenic mice. The ultimate proof that RANK expressed on
preosteoclastic cells was the sole receptor on these cells for
RANKL came with the demonstration that RANK knockout
mice had profound osteopetrosis due to an absence of oste-
oclasts (28). Moreover, osteoclastogenesis could be initiated
in these mice by transfer of the RANK cDNA back into
hematopoietic precursors. Interestingly, similar to the
RANKL knockout mice, RANK knockout mice also lacked
peripheral lymph nodes and had defective B and T cell mat-
uration but differed from the RANKL knock out mice in
having normal thymic development (28).
Human RANK is a 616-amino acid peptide, with a 28-
amino acid signal peptide, an N-terminal extracellular do-
main, a short transmembrane domain of 21 amino acids, and
a large C-terminal cytoplasmic domain (22). It is expressed
primarily on cells of the macrophage/monocytic lineage,
including preosteoclastic cells, T and B cells, dendritic cells,
and fibroblasts (22, 27). The RANKL/RANK signaling path-
way has also been extensively studied in recent years. Thus,
RANK activation by RANKL is followed by its interaction
with TNF receptor-associated (TRAF) family members, ac-
tivation of nuclear factor (NF)-␬B and c-Fos, JNK, c-src, and
the serine/threonine kinase Akt/PKB (22, 27). Consistent
with these findings, mice with various components of this
signaling pathway ablated [i.e. TRAF-6 (29) or NF-␬B1/NF-
␬B2 (30)] have an osteopetrotic phenotype.
A Complete Picture of Osteoclastogenesis Emerges
The unraveling of the OPG/RANKL/RANK system,
which occurred over a span of approximately 2 yr or less,
solved a long standing unresolved question in bone biology,
namely the precise mechanisms by which preosteoblastic/
stromal cells controlled osteoclast development. Given what
we know about the process of bone remodeling, it is easy to
understand why these early osteoblastic cells need to have
5052 Endocrinology, December 2001, 142(12):5050 –5055
the critical say in whether osteoclasts are formed or not. Thus,
bone is constantly being resorbed and formed at specific sites
in the skeleton, termed basic multicellular units. The process
begins by migration of osteoclasts to these sites (activation),
resorption of a packet of bone by these cells, a reversal phase
characterized by apoptosis of the osteoclasts, followed by a
phase of bone formation by newly formed osteoblasts. As
such, it makes sense that the critical, initial step in this pro-
cess, the development of osteoclasts, should be under the
control of preosteoblastic/stromal cells: this ensures that the
processes of bone resorption and formation will be tightly
coupled, allowing for a wave of bone formation to follow
each cycle of bone resorption, thus maintaining skeletal in-
tegrity. Further coupling between osteoblastogenesis and
osteoclastogenesis is ensured by the fact that the osteoblast
differentiation factor, cbfa1, is necessary for adequate ex-
pression of the osteoclast differentiation factor, RANKL, on
the surface of preosteoblastic/stromal cells (31).
Figure 1 summarizes the current picture of the control of
osteoclastogenesis that has emerged in the post OPG/
RANKL/RANK era. RANKL, expressed on the surface of
preosteoblastic/stromal cells, binds to RANK on the oste-
oclastic precursor cells. M-CSF, which binds to its receptor,
c-Fms, on preosteoclastic cells, appears to be necessary for
osteoclast development because it is the primary determi-
nant of the pool of these precursor cells (32). RANKL, how-
ever, is critical for the differentiation, fusion into multinu-
cleated cells, activation, and survival of osteoclastic cells.
OPG puts a brake on the entire system by blocking the effects
of RANKL. A number of proresorptive cytokines, such as
TNF-␣ and IL-1, modulate this system primarily by stimu-
lating M-CSF production (thereby increasing the pool of
preosteoclastic cells) and by directly increasing RANKL ex-
pression (33). In addition, a number of other cytokines and
hormones, such as TGF-␤ (increased OPG production) (34),
PTH (increased RANKL/decreased OPG production) (35),
FIG. 1. Current understanding of
preosteoblastic/stromal cell regulation
of osteoclastogenesis. See text for de-
tails.
Khosla • Minireview
1,25-dihydroxyvitamin D3 (increased RANKL production)
(36), glucocorticoids (increased RANKL/decreased OPG
production) (37), and estrogen (increased OPG production)
(38, 39) exert their effects on osteoclastogenesis by regulating
osteoblastic/stromal cell production of OPG and RANKL.
However, not all regulation of the osteoclast is exclusively
via the osteoblast because calcitonin acts directly on oste-
oclastic cells (40), and estrogen has been shown to induce
apoptosis of osteoclasts (41) as well as inhibit osteoclast dif-
ferentiation by interfering with RANK signaling, principally
RANKL-induced JNK activation and c-Jun activity and ex-
pression (42, 43). Moreover, TGF-␤ can also stimulate RANK
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expression on preosteoclastic cells, and thus enhance oste-
oclastic sensitivity to RANKL (44).
Recent studies have also found that the ability of preos-
teoblastic/stromal cells to support osteoclast development is
lost rapidly during differentiation down the osteoblast path-
way, due principally to down-regulation of RANKL and
increased OPG production (45). Again, this makes eminent
sense in terms of the basic multicellular unit, because
whereas early osteoblastic cells in the marrow orchestrate the
process of osteoclast development, it would clearly be coun-
terproductive for the mature osteoblastic cells laying down
osteoid on the bone surface to at the same time be stimulating
osteoclast development, which would destroy the work they
have just completed.
Implications for the Pathogenesis and Treatment of
Disorders of Bone Metabolism
As with any breakthrough in basic science, the onus rap-
idly falls on the clinical investigator to translate the findings
into a better understanding of disease as well as potentially
new treatment approaches. This process has just begun for
the OPG/RANKL/RANK system and will likely accelerate
as the tools to assess gene expression in smaller and smaller
Khosla • Minireview
amounts of biological samples with greater reliability con-
tinue to evolve.
The disorders most clearly related to alterations in this
system are familial expansile osteolysis, a rare autosomal
dominant disorder characterized by focal areas of enhanced
bone resorption, and familial Paget’s disease, both of which
are due to mutations in the signal peptide region of the
RANK protein (46). These mutations may lead to an accu-
mulation of defective RANK translation products in the se-
cretion pathway, resulting perhaps in receptor self-associa-
tion and increased constitutive RANK signal transduction.
The role of the OPG/RANKL/RANK system in the patho-
genesis of more common disorders, such as postmenopausal
or age-related osteoporosis, remains controversial. As noted
earlier, estrogen does increase OPG production by osteo-
blastic (38) and marrow stromal cells (39). However, serum
OPG levels are, if anything, higher in postmenopausal
women with osteoporosis and increased bone turnover (47),
perhaps as a homeostatic mechanism limiting their more
rapid bone loss. In addition, although OPG production by
marrow stromal cells appears to decline with age (48), serum
OPG levels have consistently been found to increase with age
in women and in men (47).
Although depressed bone formation is the major abnor-
mality in glucocorticoid-induced osteoporosis (50), bone re-
sorption is often increased or at the least, inappropriately
normal for the depressed level of bone formation (50). Be-
cause glucocorticoids are potent inhibitors of OPG produc-
tion and also stimulate RANKL levels in osteoblastic cells
(37), this decrease in the OPG/RANKL ratio may well ac-
count for an enhanced ability of preosteoblastic cells (re-
duced in number as they may be) to support osteoclast de-
velopment, leading to the observed marked imbalance
between bone formation and resorption and rapid bone loss
in this condition.
As noted earlier, PTH increases RANKL and decreases
OPG expression by osteoblastic cells, leading to a net cata-
bolic effect on bone (35). However, intermittent PTH clearly
has anabolic effects on bone, and recent data indicate that, in
contrast to continuous exposure, intermittent exposure of
marrow stromal cells to PTH does not lead to a significant
alteration in the OPG/RANKL ratio, while still stimulating
markers of bone formation (50). Thus, differential effects of
PTH on the OPG/RANKL system, depending on whether it
is administered continuously or intermittently, may well ex-
plain the catabolic vs. anabolic effects of PTH on bone.
In addition to the RANK activating mutations in familial
Paget’s disease, bone marrow stromal cells in Paget’s disease
have been shown to have enhanced RANKL expression, and
preosteoclastic cells from affected lesions have increased sen-
sitivity to RANKL (51). This combination of abnormalities
may explain, at least in part, the increased numbers of os-
teoclasts in Pagetic bone. Finally, activated T cells (as in
rheumatoid arthritis) have increased levels of RANKL ex-
pression (52) and RANKL, in the presence of M-CSF, can
induce synovial macrophages to differentiate into osteoclas-
tic bone-resorbing cells (53), thus potentially leading to the
periarticular bone loss commonly seen in various forms of
inflammatory arthritis.
OPG or other components of this system may also have
Endocrinology, December 2001, 142(12):5050 –5055 5053
therapeutic utility in conditions associated with accelerated
bone resorption, including skeletal metastases from multiple
myeloma or other tumors, and postmenopausal osteoporo-
sis. Indeed, OPG has been shown to block skeletal destruc-
tion and pain in a mouse model of sarcoma-induced bone
destruction (54). In addition, a RANK-Fc fusion protein was
effective in suppressing bone resorption and hypercalcemia
in a murine model of humoral hypercalcemia of malignancy
due to xenografts of human lung cancer (55). Finally, a single
dose of a OPG-Fc fusion protein resulted in a profound (by
up to 80%) and sustained (for up to 3 wk) suppression of bone
resorption in postmenopausal women (56). However,
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whether these approaches will translate into viable new ther-
apies for these disorders or whether alternate approaches
(such as the development of small molecules to locally reg-
ulate OPG/RANKL production in the bone microenviron-
ment) will be needed remains to be seen.
Conclusions
The pace of developments resulting in an unraveling of the
OPG/RANKL/RANK system over the past few years, lead-
ing to a nearly complete understanding of osteoblastic reg-
ulation of osteoclastogenesis, is truly breathtaking. In addi-
tion to providing fundamental insights in bone biology, these
events have identified an entirely new set of candidate fac-
tors that may be involved in the pathogenesis of a number
of rare and common metabolic bone diseases. Although only
time will tell whether these advances will translate into new
therapeutic approaches, clearly the detailed characterization
of this system has opened up entirely new areas for basic
and clinical investigation in bone biology and diseases,
respectively.
Acknowledgments
I would like to thank Drs. B. L. Riggs and T. C. Spelsberg for helpful
suggestions and comments.
Received August 21, 2001. Accepted August 31, 2001.
Address all correspondence and requests for reprints to: Sundeep
Khosla, M.D., Mayo Clinic, 200 First Street SW, 5-194 Joseph, Rochester,
Minnesota 55905. E-mail: khosla.sundeep@mayo.edu.
This work was supported by Research Grant AG04875 from the
National Institute on Aging, United States Public Health Service.
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