REVIEW

Regulators of Osteoclast Differentiation and Cell–Cell Fusion
Takeshi Miyamoto1–4
1Department of Orthopedic Surgery, School of Medicine, Keio University, Tokyo, Japan
2Keio Kanrinmaru Project, School of Medicine, Keio University, Tokyo, Japan

3Department of Integrated Bone Metabolism and Immunology, School of Medicine, Keio University, Tokyo, Japan
4Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency,

Saitama, Japan(Received for publication on October 10, 2010)(Revised for publication on March 17, 2011)(Accepted for publication on April 21, 2011)

Osteoclasts are multinuclear giant cells derived from osteoclast/macrophage/dendritic cell common
progenitor cells. The most characteristic feature of osteoclasts is multinucleation resulting from cell–
cell fusion of mononuclear osteoclasts. Osteoclast cell–cell fusion is considered essential for re-organization of the cytoskeleton, such as the actin-ring and ruffled boarder to seal the resorbing area and tosecret protons, respectively, to resorb bone; the fusion process is thus critical for osteoclast function.
Various molecules, such as E-cadherin and macrophage fusion receptor (MFR), have been identified as
regulators of osteoclast or macrophage cell–cell fusion. Laboratory production of osteoclasts used to be
performed in a co-culture of osteoclast progenitors with osteoblastic cells, but recent advances in theidentification of nuclear factor of kappa B ligand (RANKL) enabled the isolation of osteoclast-specificmolecules involving osteoclast cell–cell fusion and differentiation regulators from purified osteoclast
mRNA, since osteoclasts can be formed without osteoblasts. The essential cell–cell fusion regulator,
dendritic cell-specific transmembrane protein (DC-STAMP), was isolated by a cDNA subtractive screen
between mononuclear macrophages and RANKL-induced multinuclear osteoclasts. The cell–cell fusionof osteoclasts and foreign body giant cells (FBGCs) was completely abrogated in DC-STAMP-deficient
mice in vivo and in vitro. Bone resorbing activity was significantly reduced but was still detected in
DC-STAMP-deficient osteoclasts. DC-STAMP expression is positively regulated by two transcriptional factors: nuclear factor of activated T cells 1 (NFATc1) and c-Fos, both of which are essential for
osteoclast differentiation. Furthermore, a novel osteoclastogenesis-regulating pathway involving two
transcriptional repressors [B cell lymphoma 6 (Bcl6) and B lymphocyte-induced maturation protein 1(Blimp1)] under RANKL stimulation has been discovered. The expression of osteoclastic genes such as
DC-STAMP, NFATc1, and Cathepsin K, as well as osteoclast differentiation, was inhibited by Bcl6. Bcl6deficient mice showed enhanced osteoclastogenesis and reduced bone mass, whereas osteoclast-specificBlimp1-conditional knockout mice showed elevated Bcl6 expression, osteoclastic gene expression, andosteoclast differentiation and increased bone mass. In this review, recent advances in our understanding of the regulators of osteoclast differentiation and cell–cell fusion are discussed. (Keio J Med 60 (4): 101–105, December 2011)

Keywords: osteoclasts, DC-STAMP, Blimp1, Bcl6

Introduction
Osteoclasts are unique bone-resorbing cells, and theloss of osteoclast differentiation or function seen in osteopetrotic mice (such as op/op mice), c-Fos-deficient mice,and receptor activator of nuclear factor kappa B ligand

Reprint requests to: Takeshi Miyamoto, MD, Department of Orthopedic Surgery, School of Medicine, Keio University, 35 Shinano-machi,
Shinjuku-ku, Tokyo 160-8582, Japan, E-mail: miyamoto@z5.keio.jp
Copyright © 2011 by The Keio Journal of Medicine
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(RANKL)-deficient mice results in the abrogation ofbone resorption, which leads to lack of bone marrow andthe absence of tooth eruption.1–3 [RANKL is also known
as the osteoclast differentiation factor (ODF), osteoprotegerin ligand (OPGL), and tumor necrosis factor [TNF]related activation-induced cytokine (TRANCE).6–9] In
contrast, elevated-osteoclast differentiation or function
seen in osteoporosis and metastatic bone cancer patientsresults in reduced bone mass and bone destruction, respectively.4,5 Thus, it is necessary to understand themechanisms of osteoclastogenesis to control bone massin osteoporosis and metastatic bone cancer patients.
Until the identification of RANKL, osteoclastogenesis
had been induced in the laboratory in a co-culture systemwith osteoclast progenitor cells and osteoblastic cells under the stimulation of osteotropic factors, such as vitamin
D3.10 The identification of RANKL enabled the induction of osteoclastogenesis without osteoblastic cells in thepresence of RANKL and M-CSF. An osteoclast culture
system was established by isolating osteoclast progenitor
cells using flow cytometric sorting and cultivation in thepresence of RANKL and M-CSF11,12; in this way, various
osteoclastic factors were isolated (Fig. 1).
Identification of DC-STAMP, which is essential forcell–cell fusion in osteoclasts and FBGCs
Osteoclasts and macrophages are derived from common progenitors in the presence of M-CSF plus RANKLand M-CSF alone, respectively.13 M-CSF-induced macrophages are mononuclear cells, whereas M-CSF plus
RANKL-induced osteoclasts are multinuclear cells. A
cell–cell fusion regulator, DC-STAMP, was isolated by acDNA subtractive screen between osteoclasts and macrophages.14 DC-STAMP is a seven-transmembrane protein
and has been isolated from dendritic cells, IL-4-induced
macrophages, and osteoclasts.15–17 Interestingly, although
the expression of osteoclast differentiation markers was
normal, multinucleation of osteoclasts was completely
abrogated in DC-STAMP-deficient mice both in vivo
and in vitro, indicating that DC-STAMP is specificallyrequired for osteoclast cell–cell fusion rather than differentiation.14 Similarly, the cell–cell fusion of FBGCs,
which are also derived from common progenitor cells of
osteoclasts and macrophages, was completely abrogatedin DC-STAMP-deficient mice.14,18 Thus, DC-STAMP isessential for cell–cell fusion of both osteoclasts and FBGCs.
Role of osteoclast cell–cell fusion in bone resorption
and bone homeostasis
The absence of cell–cell fusion in osteoclasts resulted
in the severe reduction of bone-resorbing activity, which
in turn increased bone mass in DC-STAMP-deficient

Miyamoto T: Molecular Understanding of Osteoclastogenesis

mice.14 Thus, DC-STAMP plays a role in regulating bone
resorbing efficiency and physiological bone mass. Indeed,
DC-STAMP-overexpressing transgenic (DC-STAMPTg) mice showed accelerated osteoclast bone-resorbing
activity and reduced bone mass.19 Interestingly, although
DC-STAMP is not expressed in osteoblasts, osteoblastic
activity is upregulated in DC-STAMP-deficient mice, but
it is downregulated in DC-STAMP Tg mice,19 suggesting that DC-STAMP likely regulates osteoblastic activity
through osteoclast cell–cell fusion. Thus, DC-STAMP
is a bone-remodeling factor regulating both osteoclasts
and osteoblasts. A similar phenomenon was reported in
v-ATPase V0 subunit d2 (ATP6v0d2)-deficient mice; in
addition, severe inhibition of osteoclast cell–cell fusion,as well as increased osteoblastic activity, was detectedin ATP6v0d2-deficient mice.20 Thus, osteoclast cell–cellfusion might be critically involved in bone homeostasis.
Regulation of cell–cell fusion in osteoclasts
Identification of RANKL further supports the isolationof the master transcriptional regulator of osteoclastogenesis, nuclear factor of activated T cells 1 (NFATc1), sinceosteoclasts could be formed without osteoblastic cells,
and osteoclast-specific molecules have been isolated.21
Osteoclastogenesis is reportedly regulated by NFATc1
and AP1 cooperatively under RANKL stimulation. Since
the expression of DC-STAMP and ATPv0d2 was regulated by NFATc1,18,22 osteoclast cell–cell fusion was induced along with the differentiation of osteoclasts.
Identification of a novel osteoclast differentiation pathway
As described above, NFATc1 is required for osteoclastogenesis and, interestingly, auto-amplification of
NFATc1, i.e., NFATc1-dependent NFATc1 induction, hasbeen induced in osteoclasts.23 Thus, inhibiting NFATc1
was predicted to result in increased bone mass by suppressing osteoclast formation. NFAT inhibitor FK506
has been utilized clinically as an immune-suppressor fororgan transplantation; however, reduced bone mass in
FK506-treated patients as well as in mice has been observed24; this was due to the inhibition of bone formation
by FK506.24 FK506 inhibits NFAT-family molecules, and
NFATs are critically involved in regulating osteoblastogenesis and, although osteoclastogenesis is strongly inhibited by FK506, osteoblastogenesis is more strongly
suppressed by FK506, which results in reduced bonemass.24 Thus, although NFATc1-activating pathways havebeen extensively identified,25 other osteoclastogenesisregulating pathways are sought as they might be potential
therapeutic targets because inhibiting NFATc1 resulted indecreased bone mass.
In contrast to the positive regulators of osteoclastogen-Keio J Med 2011; 60 (4): 101–105

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Fig. 1 Strategy for isolating RANKL target genes, including cell–cell fusion regulators, by a DNA subtractive screen between multinuclear osteoclasts induced by M-CSF plus RANKL and mononuclear macrophages induced by M-CSF alone.

Fig. 2 The Blimp1-Bcl6-osteoclastic gene axis is critical for regulating osteoclastogenesis and bone homeostasis.
NFATc1 is crucial as a positive regulator for osteoclastogenesis under RANKL stimulation. In contrast, Blimp1 and Bcl6 are bothtranscriptional repressors and are also critical regulators for osteoclast differentiation. Blimp1 inhibits the expression of Bcl6, which
inhibits osteoclastic genes, such as DC-STAMP, Cathepsin K, and NFATc1.

esis such as AP1 and NFATc1, the negative regulatorsof osteoclast differentiation have not been fully characterized. B cell lymphoma 6 (Bcl6) is a transcriptional
repressor that was identified as being highly expressedin osteoclast progenitors but was significantly downregulated during osteoclastogenesis by RANKL.26 Bcl6
was shown to directly bind to DC-STAMP promoter andbecame dissociated upon RANKL stimulation.26 Thus
DC-STAMP expression is a good indicator for analyzing

regulators of osteoclast differentiation. Similarly, Bcl6
bound to Cathepsin K promoter and NFATc1 promoter in
the absence of RANKL and became dissociated in thepresence of RANKL; thus, suppression of Bcl6 during
differentiation is required for appropriate osteoclastogenesis and regulation of bone homeostasis. Lack of Bcl6
results in hyper-osteoclast differentiation, which in turnleads to loss of bone mass.26 Thus, osteoclastogenesis
and bone homeostasis are regulated in a delicate balance

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Miyamoto T: Molecular Understanding of Osteoclastogenesis

Fig. 3 Osteoclastogenesis and bone homeostasis are regulated by positive and negative regulators.
Positive regulators of osteoclastogenesis are calcium and the immunoreceptor tyrosine-based activation motif (ITAM) signal, which
activate NFATc1, a master positive regulator of osteoclastogenesis. Negative regulators of osteoclastogenesis are Bcl6, IRF8, andMafb.

between NFATc1-induced positive regulation and Bcl6induced negative regulation of osteoclast differentiation.
Moreover, Bcl6 expression in osteoclasts was further
negatively regulated by another transcriptional repressor,
Blimp1 (Fig. 2).26,27 Lack of Blimp1 in osteoclasts resulted in loss of Bcl6 suppression, which in turn inhibited
osteoclast differentiation in the presence of RANKL andincreased bone mass.26 Thus, the negative axis involving
Blimp1-Bcl6 is a novel pathway regulating osteoclast differentiation and bone homeostasis.
To date, other negative regulators of osteoclast differentiation, such as interferon regulatory factor 8 (IRF8)
and v-maf musculoaponeurotic fibrosarcoma oncogene
family protein B (Mafb), have been identified,28,29 andsuppression of these molecules is also required for appropriate osteoclastogenesis. Further studies are needed toclarify the mechanisms of osteoclastogenesis regulation
by positive and negative regulators.
Future Directions
Bone remodeling is regulated in a coupled manner by
osteoclasts and osteoblasts. Indeed, increased bone resorption by osteoclasts has been observed, but accelerated osteoblastic activity has also been detected in osteoporosis patients, indicating that both osteoclast andosteoblast activities are accelerated. Meanwhile, inhibited osteoclast activity by bisphosphonate also results ininhibited osteoblastic activity, and thus both osteoclast

and osteoblast activities are inhibited. This means that
osteoclast bone resorption reduces bone mass, but is also
required for subsequent bone formation by osteoblasts.
Interestingly, bone remodeling in DC-STAMP-deficientand ATP6v0d2-deficient mice is regulated in an uncoupled manner, and inhibited osteoclast activity and elevated osteoblast activity were seen in these mice, which inturn increased bone mass. Thus, osteoclast cell–cell fusion is considered a therapeutic target for bone diseases
such as osteoporosis.
In addition, osteoclast-specific Blimp1-conditional
knockout mice showed inhibited osteoclastogenesis andincreased bone mass.28,29 Osteoclastogenesis and bonehomeostasis are controlled in a delicate balance between
osteoclast-positive regulators, such as NAFTc1, and negative regulators, such as Bcl6 (Fig. 3). Since inhibition of
NFATc1 fails to increase bone mass and rather reducesbone mass, Blimp1 is an interesting therapeutic target forbone diseases. Further studies are required to identify potential therapies that increase bone mass in a more physiological manner in the future.
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