Birth and Death of Bone Cells: Basic Regulatory Mechanisms

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Endocrine Reviews 21(2): 115–137
Copyright © 2000 by The Endocrine Society
Printed in U.S.A.
Birth and Death of Bone Cells: Basic Regulatory
Mechanisms and Implications for the Pathogenesis and
Treatment of Osteoporosis
STAVROS C. MANOLAGAS
Division of Endocrinology & Metabolism, Center for Osteoporosis & Metabolic Bone Diseases,
University of Arkansas for Medical Sciences, and the Central Arkansas Veterans Healthcare System,
Little Rock, Arkansas 72205, USA
ABSTRACT
The adult skeleton regenerates by temporary cellular structures
that comprise teams of juxtaposed osteoclasts and osteoblasts and
replace periodically old bone with new. A considerable body of evidence accumulated during the last decade has shown that the rate of
genesis of these two highly specialized cell types, as well as the
prevalence of their apoptosis, is essential for the maintenance of bone
homeostasis; and that common metabolic bone disorders such as osteoporosis result largely from a derangement in the birth or death of
these cells. The purpose of this article is 3-fold: 1) to review the role
and the molecular mechanism of action of regulatory molecules, such
as cytokines and hormones, in osteoclast and osteoblast birth and
apoptosis; 2) to review the evidence for the contribution of changes in
bone cell birth or death to the pathogenesis of the most common forms
of osteoporosis; and 3) to highlight the implications of bone cell birth
and death for a better understanding of the mechanism of action and
efficacy of present and future pharmacotherapeutic agents for osteoporosis. (Endocrine Reviews 21: 115–137, 2000)
I. Introduction
II. Physiological Bone Regeneration
A. Remodeling by the basic multicellular unit (BMU)
III. Osteoblastogenesis and Osteoclastogenesis
A. Growth factors and their antagonists
B. Cytokines
C. Systemic hormones
D. Adhesion molecules
IV. Reciprocal Relationship Between Osteoblastogenesis
and Adipogenesis
V. Serial and Parallel Models of Osteoblast and Osteoclast
Development
VI. Function of the Mature Cells
A. Osteoblasts
B. Osteocytes
C. Lining cells
D. Osteoclasts
VII. Death of Bone Cells by Apoptosis
VIII. Regulation of Bone Cell Proliferation and Activity
IX. Pathogenesis of Osteoporosis
A. Sex steroid deficiency
B. Senescence
C. Glucocorticoid excess
X. Pharmacotherapeutic Implications of Osteoblast and
Osteocyte Apoptosis
A. Intermittent PTH administration
B. Bisphosphonates and calcitonin
C. Novel pharmacotherapeutic strategies
XI. Summary and Conclusions
I. Introduction
And Athena lavished a marvelous splendor on the prince so that
all the people gazed in wonder as he came forward. The elders
making way as he took his father’s seat. The first to speak was an
old lord, Aegyptius, stooped with age, who knew the world by
heart.
Homer, the Odyssey: translation by Robert Fagles
L
OSS OF height (stooping), Dowager’s hump, and kyphosis are some of the most visible signs of old age in
humans. The primary reason for these involutional changes
is a progressive loss of bone mass that affects the axial (primarily trabecular) as well as the appendicular (primarily
cortical) skeleton. Loss of bone mass, along with microarchitectural deterioration of the skeleton, leads to enhanced
bone fragility and increased fractures—the bone disease
known as osteoporosis (1). Both men and women start losing
bone in their 40s. However, women experience a rapid phase
of loss during the first 5–10 yr after menopause, due to the
loss of estrogen (2). In men this phase is obscure, since there
is only a slow and progressive decline in sex steroid production; hence, the loss of bone in men is linear and slower
(3). In addition to losing bone faster at the early postmenopausal years, women also accumulate less skeletal mass than
men during growth, particularly in puberty, resulting in
smaller bones with thinner cortices and smaller diameter.
Consequently, the incidence of bone fractures is 2- to 3-fold
higher in women as compared with men (4).
In addition to sex steroid deficiency and the aging process
Address reprint requests to: Stavros C. Manolagas, M.D., Ph.D.,
Center for Osteoporosis & Metabolic Bone Diseases, Division of
Endocrinology & Metabolism, University of Arkansas Medical School,
4301 West Markam Street, Little Rock, Arkansas 72205 USA. E-mail:
[email protected]
*Research from the author’s laboratory described in this review was
supported by the NIH (P01-AG13918, R01-AR43003), the Department of
Veterans Affairs (merit grant and a research enhancement award program, REAP), and the 1999 Allied Signal Award for Research on Aging.
115
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MANOLAGAS
itself, loss of bone mass is accentuated when several other
conditions are present. The most common are chronic glucocorticoid excess (5), particularly its iatrogenic form, hyperthyroidism as well as inappropriately high T4 replacement, alcoholism,
prolonged immobilization, gastrointestinal disorders, hypercalciuria, some types of malignancy, and cigarette smoking (6).
Bone loss and eventually fractures are the hallmarks of
osteoporosis, regardless of the underlying cause or causes.
The bone loss associated with normal aging in women has
been divided into two phases: one that is due to menopause
and one that is due to aging and affects men as well (7, 8).
In elderly women these two phases eventually overlap, making it difficult to distinguish the effect of sex steroid deficiency from the effect of the aging process itself. The effect
of the aging process itself is also frequently obscured because
of secondary hyperparathyroidism (9), resulting from impaired calcium absorption from the intestine with advancing
age (⬎75 yr old). The bone loss that is due to glucocorticoid
excess shares several features with the bone loss due to
senescence, but also has unique features of its own. Nonetheless, as is the case with the other types of bone loss, the
heterogeneity of the underlying conditions, some of which
(e.g., postmenopausal state, rheumatoid arthritis, etc.) independently contribute to skeletal deterioration, can distort the
clinical and histological picture (10). Irrespective of the overlap, it is important to recognize that the pathogenetic mechanisms are quite distinct in the various forms of osteoporosis
and that sex hormone deficiency and aging have independent effects.
During the last few years, there have been significant advances in our understanding of the pathogenetic mechanisms responsible for the bone loss associated with sex steroid deficiency, old age, and glucocorticoid excess. All these
conditions do not cause loss of bone mass by turning on a
completely new process. Instead, they cause a derangement
in the normal process of bone regeneration. Therefore, to
understand the pathogenesis of osteoporosis and rationalize
its treatment, one must first appreciate the basic principles of
physiological bone regeneration.
II. Physiological Bone Regeneration
The skeleton is a highly specialized and dynamic organ
that undergoes continuous regeneration. It consists of highly
specialized cells, mineralized and unmineralized connective
tissue matrix, and spaces that include the bone marrow cavity, vascular canals, canaliculi, and lacunae. During development and growth, the skeleton is sculpted to achieve its
shape and size by the removal of bone from one site and
deposition at a different one; this process is called modeling.
Once the skeleton has reached maturity, regeneration continues in the form of a periodic replacement of old bone with
new at the same location (11). This process is called remodeling and is responsible for the complete regeneration of the
adult skeleton every 10 yr. The purpose of remodeling in the
adult skeleton is not entirely clear, although in bones that are
load bearing, remodeling most likely serves to repair fatigue
damage and to prevent excessive aging and its consequences.
Hence, the most likely purpose of bone remodeling is to
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prevent accumulation of old bone. Remodeling, with positive
balance, does occur in the growing skeleton as well. Its purpose, quite different from those proposed for the adult skeleton, is to expand the marrow cavity while increasing trabecular thickness (12).
A. Remodeling by the basic multicellular unit (BMU)
Removal of bone (resorption) is the task of osteoclasts.
Formation of new bone is the task of osteoblasts. Bone resorption and bone formation, however, are not separate,
independently regulated processes. In the uninjured adult
skeleton, all osteoclasts and osteoblasts belong to a unique
temporary structure, known as a basic multicellular unit or
BMU (13). Although during modeling one cannot distinguish
anatomical units analogous to BMU per se, sculpting of the
growing skeleton requires spatial and temporal orchestration of the destination of osteoblasts and osteoclasts, albeit
with different rules and coordinates to those operating in the
BMU of the remodeling skeleton. The BMU, approximately
1–2 mm long and 0.2– 0.4 mm wide, comprises a team of
osteoclasts in the front, a team of osteoblasts in the rear, a
central vascular capillary, a nerve supply, and associated
connective tissue (13). In healthy human adults, 3– 4 million
BMUs are initiated per year and about 1 million are operating
at any moment (Table 1). Each BMU begins at a particular
place and time (origination) and advances toward a target,
which is a region of bone in need of replacement, and for a
variable distance beyond its target (progression) and eventually comes to rest (termination) (10). In cortical bone, the
BMU travels through the bone, excavating and replacing a
tunnel. In cancellous bone, the BMU moves across the trabecular surface, excavating and replacing a trench. In both
situations, the cellular components of the BMUs maintain a
well orchestrated spatial and temporal relationship with
each other. Osteoclasts adhere to bone and subsequently
remove it by acidification and proteolytic digestion. As the
BMU advances, osteoclasts leave the resorption site and osteoblasts move in to cover the excavated area and begin the
process of new bone formation by secreting osteoid, which
is eventually mineralized into new bone.
The lifespan of the BMU is 6 –9 months; much longer than
the lifespan of its executive cells (Table 1). Therefore, continuous supply of new osteoclasts and osteoblasts from their
respective progenitors in the bone marrow is essential for the
origination of BMUs and their progression on the bone surTABLE 1. Vital statistics of adult bone remodelinga
●
●
●
●
●
●
Lifespan of BMU ⬃6 –9 months
Speed ⬃25 ␮m/day
Bone volume replaced by a single BMU ⬃0.025 mm3
Lifespan of osteoclasts ⬃2 weeks
Lifespan of osteoblasts (active) ⬃3 months
Interval between successive remodeling events at the same
location ⬃2–5 years.
● Rate of turnover of whole skeleton ⬃10% per yearb
a
From A. Michael Parfitt (13)
The 10% per year approximation for the entire skeleton is based
on an average 4% turnover per year in cortical bone, which represents
roughly 75% of the entire skeleton; and an average 28% turnover per
year in trabecular bone, which represents roughly 25% of the skeleton
(0.75 ⫻ 4 ⫽ 3 and 0.25 ⫻ 28 ⫽ 7; 3 ⫹ 7 ⫽ 10).
b
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BIRTH AND DEATH OF BONE CELLS
face. Consequently, the balance between the supply of new
cells and their lifespan are key determinants of the number
of either cell type in the BMU and the work performed by
each type of cells and are critical for the maintenance of bone
homeostasis.
III. Osteoblastogenesis and Osteoclastogenesis
Both osteoblasts and osteoclasts are derived from precursors originating in the bone marrow. The precursors of osteoblasts are multipotent mesenchymal stem cells, which
also give rise to bone marrow stromal cells, chondrocytes,
muscle cells, and adipocytes (14 –16), whereas the precursors
of osteoclasts are hematopoietic cells of the monocyte/macrophage lineage (17, 18). Long before these cells could be
cultured, the existence of multipotent mesenchymal stem
cells was suspected (19), based on the evidence that fibroblastic colonies formed in cultures of adherent bone marrow
cells can differentiate, under the appropriate stimuli, into
each of the above mentioned cells; these progenitors were
named colony forming unit fibroblasts (CFU-F). When
CFU-F are cultured in the presence of ␤-glycerophosphate
and ascorbic acid, the majority of the colonies form a mineralized bone nodule; these bone-forming colonies are
known as CFU-osteoblast (CFU-OB) (16). Osteoblast progenitors may originate not only from stromal mesenchymal progenitors of the marrow, but also pericytes — mesenchymal
cells adherent to the endothelial layer of vessels (20).
Whereas osteoclast precursors reach bone from the circulation, osteoblast precursors most likely reach bone by migration of progenitors from neighboring connective tissues.
The development and differentiation of osteoblasts and
osteoclasts are controlled by growth factors and cytokines
produced in the bone marrow microenvironment as well as
adhesion molecules that mediate cell-cell and cell-matrix
interactions. Several systemic hormones as well as mechanical signals also exert potent effects on osteoclast and osteoblast development and differentiation. Although many details remain to be established concerning the operation of this
network, a few themes have emerged (21). First, several of the
growth factors and cytokines control each other’s production
in a cascade fashion and, in some instances, form positive and
negative feedback loops. Second, there is extensive functional redundancy among them. Third, some of the same
factors are capable of influencing the differentiation of both
osteoblasts and osteoclasts. Fourth, systemic hormones influence the process of osteoblast and osteoclast formation via
their ability to control the production and/or action of local
mediators.
A. Growth factors and their antagonists
The only factors capable of initiating osteoblastogenesis
from uncommitted progenitors are bone morphogenetic proteins (BMPs) (22). BMPs have been long implicated in skeletal
development during embryonic life and fracture healing.
More recently, it has become apparent that BMPs, and in
particular BMP-2 and – 4, also initiate the commitment of
mesenchymal precursors of the adult bone marrow to the
osteoblastic lineage (23). BMPs stimulate the transcription of
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the gene encoding an osteoblast-specific transcription factor,
known as osteoblast specific factor 2 (Osf2) or core binding
factor a1 (Cbfa1), hereafter referred to as Cbfa1 (24). In turn,
Cbfa1 activates osteoblast-specific genes such as osteopontin,
bone sialoprotein, type I collagen, and osteocalcin. The importance of Cbfa1 for osteoblasts has been highlighted by the
evidence that knockout of the Cbfa1 gene in mice prevents
osteoblast development (25, 26). In addition to Cbfa1, BMP-4
induces a homeobox-containing gene, distal-less 5(Dlx5),
which also seems to act as a transcription factor, probably as
a heterodimer with another homeobox-containing protein
(Msx2). Like Cbfa1, Dlx5 regulates the expression of osteoblast-specific genes such as osteocalcin and alkaline phosphatase, as well as mineralization (27–29). Other factors such
as transforming growth factor ␤ (TGF␤), platelet-derived
growth factor (PDGF), insulin-like growth factors (IGFs), and
members of the fibroblast growth factor (FGF) family can all
stimulate osteoblast differentiation (30, 31). However,
whereas TGF␤, PDGF, FGF, and IGFs are able to influence the
replication and differentiation of committed osteoblast progenitors toward the osteoblastic lineage, they cannot induce
osteoblast differentiation from uncommitted progenitor
cells.
In addition to growth factors, bone cells produce proteins
that modulate the activity of growth factors either by binding
to them and thereby preventing interaction with their receptors, by competing for the same receptors, or by promoting the activity of a particular factor. For example, osteoblasts
produce several IGF-binding proteins (IGFBPs). Of these,
IGFBP-4 binds to IGF and blocks its action, whereas IGFBP-5
promotes the stimulatory effects of IGF on osteoblasts (30).
During the last few years, several proteins able to antagonize
BMP action have also been discovered. Of them, noggin,
chordin, and cerberus were initially found in the Spemann
organizer of the Xenopus embryo and shown to be essential
for neuronal or head development (32–35). Noggin and chordin inhibit the action of BMPs by binding directly and with
high affinity with the latter proteins (36, 37). Such binding is
highly specific for BMP-2 and 4, as noggin binds BMP-7 with
very low affinity and does not bind TGF␤ or IGF-I. Addition
of human recombinant noggin to bone marrow cell cultures
from normal adult mice inhibits not only osteoblast, but also
osteoclast, formation, and these effects can be reversed by
exogenous BMP-2 (23). Consistent with this evidence, BMP-2
and -4 and BMP-2/4 receptor transcripts and proteins are
found in bone marrow cultures and in bone marrow-derived
stromal/osteoblastic cell lines, as well as in murine adult
whole bone. Noggin expression has also been documented in
all these cell preparations. These findings indicate that
BMP-2 and -4 are expressed in the bone marrow in postnatal
life and serve to maintain the continuous supply of osteoblasts.
B. Cytokines
Since the early stages of hematopoiesis and osteoclastogenesis proceed along identical pathways, it is not surprising
that a large group of cytokines and colony-stimulating factors that are involved in hematopoiesis also affect osteoclast
development (38). This group includes the interleukins IL-1,
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MANOLAGAS
IL-3, IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin
M (OSM), ciliary neurotropic factor (CNTF), tumor necrosis
factor (TNF), granulocyte macrophage-colony stimulating
factor (GM-CSF), M-CSF, and c-kit ligand. As opposed to the
above mentioned cytokines that stimulate osteoclast development, IL-4, IL-10, IL-18, and interferon-␥ inhibit osteoclast
development. In the case of IL-18 the effect is mediated
through GM-CSF (39).
IL-6 has attracted particular attention because of evidence
that it plays a pathogenetic role in several disease states
characterized by accelerated bone remodeling and excessive
focal or systemic bone resorption (40). IL-6 is produced at
high levels by cells of the stromal/osteoblastic lineage in
response to stimulation by a variety of other cytokines and
growth factors such as IL-1, TNF, TGF␤, PDGF, and IGF-II
(41– 43). Binding of IL-6 or other members of the same cytokine family (IL-11, LIF, OSM) to cytokine-specific cell surface receptors (in the case of IL-6, the IL-6R␣) causes recruitment and homo- or heterodimerization of the signal
transducing protein gp130, which is then tyrosine phosphorylated by members of the Janus family of tyrosine kinases
(JAKs) (44). This event results in tyrosine phosphorylation of
several downstream signaling molecules, including members of the signal transducers and activators of transcription
(STAT) family of transcription factors (45, 46). Phosphorylated STATs, in turn, undergo homo- and heterodimerization
and translocate to the nucleus where they activate cytokineresponsive gene transcription (47). The ␣-subunit of the IL-6
receptor also exists in a soluble form (sIL-6R), but unlike most
soluble cytokine receptors, it functions as an agonist by binding to IL-6 and then interacting with membrane-associated
gp130 to stimulate JAK/STAT signaling (44). On the other
hand, the soluble form of gp130 blocks IL-6 action (48).
Alone or in concert with other agents, IL-6 stimulates
osteoclastogenesis and promotes bone resorption. The cells
that mediate the actions of the IL-6 type cytokines on osteoclast formation appear to be the stromal/osteoblastic cells,
as stimulation of IL-6R␣ expression on these cells allows
them to support osteoclast formation in response to IL-6 (49).
These findings indicate that the osteoclastogenic property of
IL-6 depends not only on its ability to act directly on hematopoietic osteoclast progenitors, but also on the activation of
gp130 signaling in the stromal/osteoblastic cells that provide
essential support for osteoclast formation. STAT3 activation
in stromal/osteoblastic cells is essential for gp130-mediated
osteoclast formation (50). Despite the effects of IL-6 on osteoclastogenesis in experimental in vitro systems, IL-6 is not
required for osteoclastogenesis in vivo under normal physiological conditions. In fact, osteoclast formation is unaffected in sex steroid-replete mice treated with a neutralizing
anti-IL-6 antibody, or in IL-6-deficient mice (51, 52). The most
likely explanation for this is that the ␣-subunit of the IL-6
receptor in bone is a limiting factor, and that both a change
in the receptor and the cytokine are required for the IL-6mediated increased osteoclastogenesis, seen in pathological
states.
IL-6 type cytokines are capable of influencing the differentiation of osteoblasts as well. Thus, receptors for these
cytokines are expressed on a variety of stromal/osteoblastic
cells, and ligand binding induces progression toward a more
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mature osteoblast phenotype, characterized by increased alkaline phosphatase and osteocalcin expression, and a concomitant decrease in proliferation (53, 54). Moreover, IL-6
type cytokines stimulate the development of osteoblasts
from noncommitted embryonic fibroblasts obtained from
12-day-old murine fetuses (55). Consistent with the in vitro
evidence, several in vivo studies have demonstrated increased bone formation in transgenic mice overexpressing
OSM or LIF (56, 57).
TGF␤ is another example of a factor affecting both bone
formation and bone resorption (58). Thus, in addition to its
ability to stimulate osteoblast differentiation, TGF␤ increases
bone resorption by stimulating osteoclast formation. Injection of TGF␤ into the subcutaneous tissue that overlies the
calvaria of adult mice causes increased bone resorption accompanied by the development of unusually large osteoclasts, as well as increased bone formation. The effects of
TGF␤ might be mediated by other cytokines involved in
osteoclastogenesis as TGF␤ can stimulate their production.
Mice lacking the TGF␤1 gene due to targeted disruption
exhibit excessive production of inflammatory cells, suggesting that this growth factor normally operates to suppress
hematopoiesis (59).
C. Systemic hormones
The two principal hormones of the calcium homeostatic
system, namely PTH and l,25-dihydroxyvitamin D3 [1,25(OH)2D3], are potent stimulators of osteoclast formation (17,
60). The ability of these hormones to stimulate osteoclast
development and to regulate calcium absorption and excretion from the intestine and kidney, respectively, are the key
elements of extracellular calcium homeostasis. Calcitonin,
the third of the classical bone-regulating hormones, inhibits
osteoclast development and activity and promotes osteoclast
apoptosis. Although the antiresorptive properties of calcitonin have been exploited in the management of bone diseases
with increased resorption, the role of this hormone in bone
physiology in humans, if any, remains questionable (61– 63).
PTH, PTH-related peptide, and 1,25-(OH)2D3 stimulate the
production of IL-6 and IL-11 by stromal/osteoblastic cells
(49, 64 – 66). Several other hormones, including estrogen, androgen, glucocorticoids, and T4, exert potent regulatory influences on the development of osteoclasts and osteoblasts
by regulating the production and/or action of several cytokines (21, 64, 67– 69).
D. Adhesion molecules
In addition to autocrine, paracrine, and endocrine signals,
cell-cell and cell-matrix interactions are also required for the
development of osteoclasts and osteoblasts (70 –72). Such
interactions are mediated by proteins expressed on the surface of these cells and are responsible for contact between
osteoclast precursors with stromal/osteoblastic cells and facilitation of the action of paracrine factors anchored to the
surface of cells that are required for bone cell development.
Adhesion molecules are also involved in the migration of
osteoblast and osteoclast progenitors from the bone marrow
to sites of bone remodeling as well as the cellular polarization
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BIRTH AND DEATH OF BONE CELLS
of osteoclasts and the initiation and cessation of osteoclastic
bone resorption. More important, for the purpose of this
review, adhesion molecules play a role in the control of
osteoblast and osteoclast development and apoptosis (73–77).
The list of adhesion molecules that influence bone cell
development and function includes the integrins, particularly ␣v␤3 and ␣2␤1, selectins, and cadherins, as well as a
family of transmembrane proteins containing a disintegrin
and metalloprotease domain (ADAMS). Each of these proteins recognizes distinct ligands. For example, some integrins
recognize a specific amino acid sequence (RGD) present in
collagen, fibronectin, osteopontin, thrombospondin, bone
sialoprotein, and vitronectin (78).
IV. Reciprocal Relationship Between
Osteoblastogenesis and Adipogenesis
The cells that comprise the bone marrow stroma can serve
several diverse functions including support of hematopoiesis
and osteoclastogenesis, fat accumulation, and bone formation (79). This functional adaptation is apparently accomplished by the plasticity of some of the stem cell progeny as
exemplified by the ability of stromal cells to convert between
the osteoblast and adipocyte phenotype. Thus, a stromal cell
type known as the Westen-Bainton cell exhibits PTH receptors and high alkaline phosphatase activity and gives rise to
osteoblasts during fetal development and in hyperparathyroidism. On the other hand, when marrow hematopoietic
activity is reduced using chemotherapeutic agents, these
cells convert into adipocytes and can support myeloid cell
production (80 – 84). Further, adipocytes isolated by limiting
dilution from cultures of rabbit bone marrow can form bone
in diffusion chamber implants (85). Conversely, addition of
certain fatty acids to cultures of osteoblastic cells causes them
to differentiate into adipocyte-like cells (86).
It is likely that interconversion of stromal cells among
phenotypes, as well as commitment to a particular lineage
with suppression of alternative phenotypes, is governed by
specific transcription factors. Indeed, Cbfa1 is required for
commitment of mesenchymal progenitors to the osteoblast
lineage. Mice that are deficient in this factor lack osteoblasts
and mineralized bone matrix (26); and expression of Cbfa1
in fibroblastic cells induces transcription of osteoblastspecific genes (24). On the other hand, CCAAT/enhancer
binding protein ␣ (C/EBP␣), C/EBP␤, and C/EBP␦, as well
as peroxisome proliferator activated receptor ␥1 (PPAR␥1)
and PPAR␥2 orchestrate adipocyte differentiation (87–90).
Introduction of C/EBP␣ in fibroblastic cells induces adipocyte differentiation (91, 92), and transfection of fibroblastic
cells with PPAR␥2 and subsequent activation with an appropriate ligand causes the development of adipocytes (93).
Using clonal cell lines isolated from the murine bone marrow, it has been demonstrated that PPAR␥2 can convert
stromal cells from a plastic osteoblastic phenotype that reversibly expresses adipocyte characteristics to terminally differentiated adipocytes. Moreover, PPAR␥2 suppresses the
expression of Cbfa1 and thereby osteoblast-specific genes
(94). Similar to the inhibitory effect of PPAR␥2 on the osteoblast phenotype, the combination of PPAR␥ and C/EBP␣
119
suppresses the muscle cell phenotype when transfected into
G8 myoblastic cells (95). Taken together, these findings
strongly suggest that PPAR␥2 plays a hierarchically dominant role in the determination of the fate of mesenchymal
progenitors, due to its ability to inhibit the expression of
other lineage-specific transcription factors. Studies with a
clonal cell line (2T3) suggest that BMP-2 induces osteoblast
or adipocyte differentiation in mesenchymal precursors, depending on whether the BMP receptor type IA or IB is activated. Therefore, BMP receptors may also play a critical role
in both the specification and reciprocal differentiation of
osteoblast and adipocyte progenitors (96).
V. Serial and Parallel Models of Osteoblast and
Osteoclast Development
Even though millions of small packets of bone are constantly remodeled, bone mass is preserved thanks to a remarkably tight balance between the amount of bone resorbed
and formed during each cycle of remodeling. In any established BMU, bone resorption and formation are happening
at the same time; new osteoblasts assemble only at sites
where osteoclasts have recently completed resorption, a phenomenon referred to as coupling, and formation begins to
occur while resorption advances. The end result is a new
packet of bone, either a cylindrical osteon or Haversian system, or a plate-like hemiosteon, that has replaced the older
bone that was removed (97).
As the BMU advances, cells are successively recruited at
each new cross-sectional location. Osteoblasts do not arrive
until the osteoclasts have moved on. However, during the
longitudinal progression of the BMU as a whole, new osteoclasts and new osteoblasts are needed simultaneously, although not at the same location. Two models of osteoblast
recruitment, a serial and a parallel (Fig. 1), can explain the
distinction between the cross-sectional and longitudinal
events during BMU progression (38). According to the serial
model, factors released from resorbed bone or the local increase in mechanical strain resulting from bone resorption,
stimulate osteoblast precursor cell proliferation and differentiation (98 –100). According to the parallel model, osteoblast and osteoclast precursor proliferation and differentiation occur concurrently in response to whatever signal
conveys the need for initiation of new BMUs, and whatever
hormone prolongs their progression (10, 38). With either
model, new osteoblasts must be directed to the right location.
Concurrent osteoblast and osteoclast production makes
teleological sense as at least one of the means of maintaining
a balance between bone formation and resorption under
normal conditions. In support of the existence of a parallel
pathway of osteoblast and osteoclast formation, it is well
established that osteoclasts cannot be formed in vitro unless
appropriate stromal cells, analogous to the bone marrow
stromal cells that support hematopoiesis, are present to provide essential support. The precise phenotype of the cells that
support osteoclast development remains unknown, but they
are clearly related to both the osteoblast and the bone marrow stromal/adipocytic lineages (101–104). Interestingly,
bone marrow-derived cells with both osteoblastic and adi-
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MANOLAGAS
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FIG. 1. Serial and parallel models of osteoblast and osteoclast development.
For explanation, please see text.
PreOC, Preosteoclast; GF, growth factors released from the matrix of
resorbed bone; preOB, osteoblast progenitors. The expressions of RANK ligand and RANK on preosteoblasts and
preosteoclasts, respectively, are depicted to indicate their critical contribution in osteoclastogenesis and thereby
the dependency of osteoclastogenesis on
preosteoblastic cells.
pogenic characteristics support the formation of osteoclasts,
but marrow-derived cells that exhibit a purely osteoblastic or
adipocytic phenotype do not (94). More to the point, noggin,
a BMP antagonist, blocks not only osteoblastogenesis but
also osteoclastogenesis in murine bone marrow cultures, indicating that commitment of mesenchymal progenitors to the
osteoblastic lineage is prerequisite for osteoclastogenesis
(23). This evidence suggests that the early less differentiated
progeny of common mesenchymal progenitors of the osteoblastic and adipocytic lineage can support osteoclast development, but more differentiated cells that have committed to
either the osteoblast or the adipocyte pathway lose this property. It is possible, but as yet untested, that the cells that
provide support for osteoclast development are a distinct
progeny of mesenchymal progenitors, which displays permanently a phenotype with mixed adipocytic/osteoblastic
characteristics, but never progresses to a terminally differentiated osteoblast or adipocyte. For convenience and lack of
a better term, the cells that support osteoclast development
are frequently referred to as stromal/osteoblastic to indicate
their similarities to both bone marrow stromal cells and osteoblasts.
In full agreement with the in vitro evidence for the dependency of osteoclast development on support by cells related to osteoblasts, mice lacking osteoblasts due to Cbfa1
deficiency also lack osteoclasts (26). In addition, marrow cells
from SAMP6 mice, a strain with defective osteoblastogenesis,
exhibit decreased osteoclastogenesis (105) and do not exhibit
the expected increase in osteoclastogenesis nor do they lose
bone after loss of sex steroids (106).
The molecular mechanism of the dependency of osteoclastogenesis on cells of the mesenchymal lineage has been elucidated during the last 2 yr with the discovery of three
proteins involved in the TNF signaling pathway (reviewed
in Ref. 107). Two of these proteins are membrane-bound
cytokine-like molecules: the receptor activator of nuclear factor-␬B (NF-␬B) (RANK) and the RANK-ligand. Other names
used in the literature for RANK are osteoprotegerin ligand
(OPG-L) and TRANCE. RANK is expressed in hematopoietic
osteoclast progenitors, while RANK-ligand is expressed in
committed preosteoblastic cells and T lymphocytes (108 –
110). RANK-ligand binds to RANK with high affinity. This
interaction is essential and, together with M-CSF, sufficient
for osteoclastogenesis. 1,25-(OH)2D3, PTH, PTHrP, gp130 activating cytokines (e.g., IL-6, IL-11), and IL-1 induce the expression of the RANK-ligand in stromal/osteoblastic cells
(50, 107). Osteoprotegerin (OPG), the third of the three proteins, unlike the other two, is a secreted disulfide-linked
dimeric glycoprotein. A hydrophobic leader peptide and
three and one-half TNF receptor-like cysteine-rich pseudorepeats characterize the amino terminus of this protein.
Unlike other members of the TNF receptor family, OPG does
not posses a transmembrane domain. OPG has very potent
inhibitory effects on osteoclastogenesis and bone resorption
in vitro and in vivo (111). Consistent with an important role
of OPG in the regulation of osteoclast formation, OPG transgenic mice develop osteopetrosis, whereas OPG knockout
mice exhibit severe osteoporosis (112). The antiosteoclastogenic property of OPG is due to its ability to act as a decoy
by binding to RANK-ligand and blocking the RANK-ligand/
RANK interaction. In addition to skeletal metabolism, the
RANK/RANK-ligand/OPG circuit may regulate several
other biological systems. Indeed, OPG is produced by many
tissues other than bone, including skin, liver, stomach, intestine, lung, heart, kidney, and placenta as well as hematopoietic and immune organs. Consistent with this, mice
deficient in RANK-ligand completely lacked lymph nodes as
well as osteoclasts (113). Moreover, OPG is also a receptor for
the cytotoxic ligand TRAIL (TNF-related apoptosis-inducing
ligand) to which it binds with high affinity and inhibits
TRAIL-mediated apoptosis in lymphocytes (114) and also
regulates antigen presentation and T cell activation (115).
Osteoblastic cells and T lymphocytes, the two cell types
that express high levels of RANK-ligand, are also the two cell
types that express high levels of the osteoblast-specific transcription factor Cbfa1 (24). More intriguingly, both the murine and human RANK-ligand genes contain two functional
Cbfa1 sites, and mutation of these sites abrogates the tran-
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BIRTH AND DEATH OF BONE CELLS
scriptional activity of the RANK-ligand gene promoter (116).
Therefore, the cell-specific expression of RANK-ligand in
cells of the stromal/osteoblastic lineage and concurrent differentiation of osteoblasts and osteoclasts might be dictated,
at least in part, by interaction between an osteoblast-specific
transcription factor and RANK-ligand. BMP 2 and -4 stimulate Cbfa1 expression. Based on these lines of evidence, it
has been postulated that the molecular underpinnings of the
control of the rate of bone regeneration and the concurrent
production of osteoclasts and osteoblasts could well be a
BMP3Cbfa13RANK-ligand gene expression cascade in
cells of the bone marrow stromal/osteoblastic lineage (117,
118). According to this hypothesis, BMPs may provide the
tonic baseline control of both processes, and thereby the rate
of bone remodeling, upon which other inputs (e.g., biomechanical, hormonal, etc.) operate.
Studies with transgenic and knockout animal models as
well as models with spontaneous genetic mutations have
identified at least three transcription factors that are required
for osteoclast differentiation: PU-1, fos, and NF-␬b. A review
of the precise role of these factors is beyond the scope of this
article, but the reader is referred to a recent excellent review
of the topic (119).
VI. Function of the Mature Cells
A. Osteoblasts
The fully differentiated osteoblasts produce and secrete
proteins that constitute the bone matrix (120). The matrix is
subsequently mineralized under the control of the same cells.
A major product of the bone-forming osteoblast is type I
collagen. This polymeric protein is initially secreted in the
form of a precursor, which contains peptide extensions at
both the amino-terminal and carboxyl ends of the molecule.
The propeptides are proteolytically removed. Further extracellular processing results in mature three-chained type I
collagen molecules, which then assemble themselves into a
collagen fibril. Individual collagen molecules become interconnected by the formation of pyridinoline cross-links,
which are unique to bone. Bone-forming osteoblasts synthesize a number of other proteins that are incorporated into the
bone matrix, including osteocalcin and osteonectin, which
constitute 40% to 50% of the noncollagenous proteins of
bone. Mice deficient in osteocalcin develop a phenotype
marked by higher bone mass and improved bone quality,
suggesting that osteocalcin functions normally to limit bone
formation without compromising mineralization (121). Conversely, mice deficient in osteonectin exhibit decreased osteoclast and osteoblast numbers and bone remodeling and
profound osteopenia, suggesting that, under normal conditions, this protein may play a role in the birth or survival of
these cells (122). Other osteoblast-derived proteins include
glycosaminoglycans, which are attached to one of two small
core proteins: PGI (or biglycan) and decorin; the latter has
been implicated in the regulation of collagen fibrillogenesis.
A number of other minor proteins such as osteopontin, bone
sialoprotein, fibronectin, vitronectin, and thrombospondin
serve as attachment factors that interact with integrins.
In addition to being the cells that produce the osteoid
121
matrix, mature osteoblasts are essential for its mineralization, the process of deposition of hydroxyapatite (123, 124).
Osteoblasts are thought to regulate the local concentrations
of calcium and phosphate in such a way as to promote the
formation of hydroxyapatite. In view of the highly ordered,
well aligned, collagen fibrils complexed with the noncollagenous proteins formed by the osteoblast in lamellar bone, it
is likely that mineralization proceeds in association with, and
perhaps governed by, the heteropolymeric matrix fibrils
themselves. Osteoblasts express relatively high amounts of
alkaline phosphatase, which is anchored to the external surface of the plasma membrane. Alkaline phosphatase has been
long thought to play a role in bone mineralization. Consistent
with this, deficiency of alkaline phosphatase due to genetic
defects leads to hypophosphatasia, a condition characterized
by defective bone mineralization (125). However, the precise
mechanism of mineralization and the exact role of alkaline
phosphatase in this process remain unclear. Bone mineralization lags behind matrix production and, in remodeling
sites in the adult bone, occurs at a distance of 8 –10 ␮m from
the osteoblast. Matrix synthesis determines the volume of
bone but not its density. Mineralization of the matrix increases the density of bone by displacing water, but does not
alter its volume.
B. Osteocytes
Some osteoblasts are eventually buried within lacunae
of mineralized matrix. These cells are termed osteocytes
and are characterized by a striking stellate morphology,
reminiscent of the dendritic network of the nervous system (126, 127). Osteocytes are the most abundant cell type
in bone: there are 10 times as many osteocytes as osteoblasts. Osteocytes are regularly spaced throughout the
mineralized matrix and communicate with each other and
with cells on the bone surface via multiple extensions of
their plasma membrane that run along the canaliculi; osteoblasts, in turn, communicate with cells of the bone
marrow stroma which extend cellular projections onto
endothelial cells inside the sinusoids. Thus, a syncytium
extends from the entombed osteocytes all the way to the
vessel wall (128) (Fig. 2). As a consequence, the strategic
location of osteocytes makes them excellent candidates for
mechanosensory cells able to detect the need for bone
augmentation or reduction during functional adaptation
of the skeleton, and the need for repair of microdamage,
and in both cases to transmit signals leading to the appropriate response; albeit this remains hypothetical (129).
Osteocytes evidently sense changes in interstitial fluid
flow through canaliculi (produced by mechanical forces)
and detect changes in the levels of hormones, such as
estrogen and glucocorticoids, that influence their survival
and that circulate in the same fluid (130 –132). Therefore,
disruption of the osteocyte network is likely to increase
bone fragility.
C. Lining cells
The surface of normal quiescent bone (i.e., bone that is
not undergoing remodeling) is covered by a 1–2-␮m thick
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MANOLAGAS
Vol. 21, No. 2
FIG. 2. Functional syncytium comprising osteocytes, osteoblasts, bone marrow stromal cells, and endothelial cells.
[Adapted from G. Marotti and reproduced with the permission of the Editor
of Journal of Clinical Investigation 104:
1363–1374, 1999 (219).
layer of unmineralized collagen matrix on top of which
there is a layer of flat and elongated cells. These cells are
called lining cells and are descendents of osteoblasts (13).
Conversion of osteoblasts to lining cells represents one of
the fates of osteoblasts that have completed their bone
forming function; another being entombment into the matrix as osteocytes. Osteoclasts cannot attach to the unmineralized collagenous layer that covers the surface of normal bone. Therefore, other cells, perhaps the lining cells,
secrete collagenase, which removes this matrix before osteoclasts can attach to bone. It has been proposed that
targeting of osteoclast precursors to a specific location on
bone depends on a “homing” signal given by lining cells;
and that lining cells are instructed to do so by osteocytes,
the only bone cells that can sense the need for remodeling
at a specific time and place (133).
vacuolar H⫹-ATPase) located in the ruffled border membrane. The protein components of the matrix, mainly collagen, are degraded by matrix metalloproteinases, and cathepsins K, B, and L are secreted by the osteoclast into the
area of bone resorption (134). The degraded bone matrix
components are endocytosed along the ruffled border within
the resorption lacunae and then transcytosed to the membrane area opposite the bone, where they are released (135,
136). Another feature of osteoclasts is the presence of high
amounts of the phosphohydrolase enzyme, tartrate-resistant
acid phosphatase (TRAPase). This feature is commonly used
for the detection of osteoclasts in bone specimens (137). Mice
deficient in TRAPase exhibit a mild osteopetrotic phenotype
(due to an intrinsic defect of osteoclastic resorptive activity)
and defective mineralization of the cartilage in developing
bones (138).
D. Osteoclasts
Mature osteoclasts are usually large (50 to 100 ␮m diameter) multinucleated cells with abundant mitochondria, numerous lysosomes, and free ribosomes. Their most remarkable morphological feature is the ruffled border, a complex
system of finger-shaped projections of the membrane, the
function of which is to mediate the resorption of the calcified
bone matrix (17, 123). This structure is completely surrounded by another specialized area, called the clear zone.
The cytoplasm in the clear zone area has a uniform appearance and contains bundles of actin-like filaments. The clear
zone delineates the area of attachment of the osteoclast to the
bone surface and seals off a distinct area of the bone surface
that lies immediately underneath the osteoclast and which
eventually will be excavated. The ability of the clear zone to
seal off this area of bone surface allows the formation of a
microenvironment suitable for the operation of the resorptive apparatus.
The mineral component of the matrix is dissolved in the
acidic environment of the resorption site, which is created by
the action of an ATP-driven proton pump (the so-called
VII. Death of Bone Cells by Apoptosis
The average lifespan of human osteoclasts is about 2
weeks, while the average lifespan of osteoblasts is 3
months (Table 1). After osteoclasts have eroded to a particular distance, either from the central axis in cortical bone
or to a particular depth from the surface in cancellous
bone, they die and are quickly removed by phagocytes
(139). The majority (65%) of the osteoblasts that originally
assembled at the remodeling site also die (140). The remaining are converted to lining cells that cover quiescent
bone surfaces or are entombed within the mineralized
matrix as osteocytes (Fig. 3A). Both osteoclasts and osteoblasts die by apoptosis, or programmed cell death, a process common to several regenerating tissues (141). As in
other tissues, bone cells undergoing apoptosis are recognized by condensation of chromatin, the degradation of
DNA into oligonucleosome-sized fragments, and the formation of plasma and nuclear membrane blebs (Fig. 4).
Eventually the cell breaks apart to form so-called apoptotic
bodies. Osteoblast apoptosis explains the fact that 50 –70%
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BIRTH AND DEATH OF BONE CELLS
123
FIG. 3. Osteoblast apoptosis and its implications. A, The average life span of a matrix forming osteoblast (⬃200 h in the mouse) is indicated
by the continuous line. The process of apoptosis represents only a small fraction of this time period. The alternative two fates of osteoblasts
are to become lining cells or osteocytes. The fraction of osteoblasts that undergo apoptosis in vivo (fApoptosis) can be estimated from a bone biopsy
specimen. The duration of the apoptosis phase that can be observed in the specimen (tApop) depends on the sensitivity of the detection method.
For example, in the case of the TUNEL technique (without CuSO4 enhancement), the TUNEL-labeled phase of apoptosis is estimated to be
approximately 2 h. In a steady state, the fraction of cells at a particular stage is the same as the corresponding fraction of time spent in that
stage. Assuming an apoptosis detection time of 2 h and a 200-h life span, a prevalence of TUNEL positive osteoblasts in the biopsy of 0.005
indicates that half of the osteoblasts die by apoptosis. B, A change in the timing and extent of osteoblast apoptosis (fApoptosis) from 50% to zero
should increase the number of osteoblasts present at the site of bone formation and thereby the work output, i.e., the amount of bone formed
by a given team of matrix-forming osteoblasts. It will also lead to an increase in the density of osteocyte apoptosis, as illustrated by the example
shown.
of the osteoblasts initially present at the remodeling site of
human bone cannot be accounted for after enumeration of
lining cells and osteocytes (142). Moreover, the frequency
of osteoblast apoptosis in vivo is such that changes in its
timing and extent could have a significant impact in the
number of osteoblasts present at the site of bone formation
(130). Osteocytes are long-lived but not immortal cells;
some die by apoptosis (132, 143, 144). Osteocyte apoptosis
could be of importance to the origination and/or progression of the BMU. Indeed, osteocytes are the only cells in
bone that can sense the need for remodeling at a specific
time and place. Moreover, osteocytes are in direct physical
contact with lining cells on the bone surface, and targeting
of osteoclast precursors to a specific location on bone
depends on a “homing” signal given by lining cells (133).
The same growth factors and cytokines that stimulate osteoclast and osteoblast development can also influence their
apoptosis. For example, TGF␤ promotes osteoclast apoptosis
while it inhibits osteoblast apoptosis. IL-6 type cytokines
have antiapoptotic effects on animal and human osteoblastic
cells (and at least in vitro they antagonize proapoptotic effects
of glucocorticoids) as well as on osteoclasts and their progenitors (54, 140, 145, 146).
VIII. Regulation of Bone Cell Proliferation and
Activity
A large body of literature suggests that growth factors,
cytokines, hormones, and drugs regulate the proliferation of
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Vol. 21, No. 2
FIG. 4. Two osteoblasts undergoing apoptosis in a section of murine cancellous
bone (TUNEL staining with toluidine
blue counterstain, ⫻630). Apoptotic osteoblasts (shown in brown) are adjacent
to an intact osteoblast (shown in blue),
on the surface of a trabecula occupying
the right lower portion of the picture in
which two intact (blue stained) osteocytes are also seen. Apoptotic osteoblasts display nuclear condensation
and fragmentation. [Photomicrograph
provided by Robert S. Weinstein, M.D.,
University of Arkansas for Medical Sciences.]
committed cells or the biosynthetic and functional activity of
the differentiated osteoblasts and osteoclasts. Hence, in addition to cell number, alterations in the functional activity of
individual cells, i.e., cell vigor, may contribute to changes in
the rate of bone resorption and formation. However, because
of the inherent difficulty in demonstrating changes in individual cell vigor in vivo, the vast majority of such literature
and its conclusions rely heavily, if not exclusively, on in vitro
experimentation. A detailed discussion of this work is beyond the scope of this review, and the reader is referred to
other articles (30, 147–151). Nonetheless, some general aspects merit discussion here, as they are important for putting
the significance of birth rate and apoptosis into a broader
perspective.
In regenerating tissues, the initial commitment of a stem
cell progeny is followed by amplification with several or
many rounds of cell division. In most tissues, division of stem
cells is infrequent, and almost all of the divisions that produce the final population of differentiated cells occur in the
so-called transit compartment (152). This notion is obscured
by the frequent practice of showing linear diagrams representing the transition from one cell type to another and
ignoring completely the amplification during the transition.
In general, terminally differentiated cells do not divide, and
an osteoblast, defined as a cell making bone matrix, and
osteoclast, as a cell resorbing bone, are in this category.
Therefore, the in vivo relevance of much of the in vitro evidence on the regulation of osteoblastic or osteoclastic cell
proliferation, using established cell lines or primary cultures
of isolated cells, must be largely confined to changes in this
transit compartment. Irrespective of whether a given regulatory factor, be that a cytokine or a hormone, influences the
initial commitment of a stem cell, or the subsequent amplification of its progeny, or both, the end result is a change in
the rate of production and therefore the number of cells
available for the execution of the biological task. For the sake
of simplicity, the terms birth, rate of birth, osteoblastogen-
esis, or osteoclastogenesis, as used in this article, implicitly
combine commitment and amplification.
The in vivo relevance of numerous reports of in vitro observations of changes in the biosynthetic activity of osteoblastic cells, e.g., changes in the level of expression of osteocalcin or alkaline phosphatase in response to a given agent,
is also a matter of conjecture. Most likely, given the nature
of commonly used in vitro cell models which, by and large,
represent preterminally differentiated cells, such observations might be more relevant to postcommitment differentiation events, than to altered activity of the fully differentiated cell. Moreover, even if some agents can alter the
function of terminally differentiated cells in short-term cultures in vitro, heretofore there is no evidence that short-term
change in the rate of collagen production or bone matrix
digestion, for example, are ultimately translated into differences in the amounts of bone matrix formed or resorbed.
In the bone literature there is considerable ambiguity
when using the term “activation.” It is important that one
distinguishes between activation as a switch from an off state
to an on state, and activation as modulation of activity of an
already active cell. Morphological evidence summarized
elsewhere does not support the notion that completely inactive osteoclasts are waiting for a stimulus to make them
active (117). However, this evidence does not address the
issue of whether there are variations in the rate of bone
matrix dissolution by individual osteoclasts after they started
work.
Studies with the widely used bone slice pit bioassay have
shown that several regulatory factors can cause a decrease or
increase in the resorptive ability of individual osteoclasts
(153–155). However, it is not clear to what extent these observations reflect a change in cell vigor as opposed to a
change in the precariously short lifespan of osteoclasts in
vitro. In estrogen deficiency, individual osteoclasts are seemingly more “active” as they dig deeper resorption cavities
often leading to trabecular perforation, but it has been con-
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BIRTH AND DEATH OF BONE CELLS
vincingly argued that this is due to delayed apoptosis (133).
In Paget’s disease osteoclasts are certainly far more aggressive than normal, perhaps as a result of their uniquely large
size and number of nuclei (156). Today, the most compelling
evidence in support of the notion that the vigor of individual
osteoclasts may not always be maximal comes from in vitro
as well as in vivo experiments with soluble RANK-ligand
(157). Specifically, it has been found that RANK-ligand acts
on mature rat osteoclasts in vitro to stimulate more frequent
cycles of resorption and induce rearrangements of the cytoskeleton. Moreover, intravenous administration of RANKligand to mice elevates the circulating concentration of
ionized calcium within 1 h. RANK-ligand has potent antiapoptotic effects on cultured osteoclasts (Ref. 158 and William Boyle, personal communication). Therefore, definitive
conclusions regarding RANK-ligand’s ability to modulate
osteoclast vigor will have to await dissection of the contribution of its effects on osteoclast survival in vivo. Similar to
osteoclasts, the bone-forming ability of osteoblasts may not
be always maximal, as suggested by the evidence that PTH
can rapidly enhance bone formation when administered by
subcutaneous injections to rats (159).
To conclude this section, it is intuitive that the amount of
bone resorbed or formed by a team of osteoclasts and osteoblasts should be a function of the total cell number as well
as the vigor of individual cells. However, whereas cell number can be quantified on bone sections from animals and
humans with conventional histomorphometric techniques,
quantification of individual cell vigor cannot. This situation
makes it difficult to judge at present whether cell vigor is or
is not a critical component in the pathogenesis of abnormal
skeletal regeneration in common acquired metabolic bone
diseases such as postmenopausal, senile, or steroid-induced
osteoporosis. For this reason and space limitations, changes
in bone cell vigor or other potential mechanisms of osteoporosis resulting from changes in extraskeletal tissues, for
example altered calcium absorption or excretion, will not be
discussed in the following section. This also reflects the author’s intention to focus on the dynamics of bone cell number, rather than a dismissal of other mechanisms.
125
IX. Pathogenesis Of Osteoporosis
From the brief discussion of the principles of physiological
bone regeneration and the role of osteoblasts and osteoclasts
in the process, it is obvious that the rate of supply of new
osteoblasts and osteoclasts and the timing of the death of
these cells by apoptosis are critical determinants of the initiation of new BMUs and/or extension or shortening of the
lifetime of existing ones. Recent advances in our understanding of the pathogenesis of the various forms of osteoporosis
have confirmed this truism by revealing that over- or undersupply of these cells relative to the need for remodeling
are the fundamental problems in all these conditions (160)
(Table 2).
A. Sex steroid deficiency
The mechanism of action of sex steroids on the skeleton is
not fully understood. At menopause (or after castration in
men), the rate of bone remodeling increases precipitously.
This fact may be explained by evidence, derived primarily
from studies in mice, that loss of sex steroids up-regulates the
formation of osteoclasts and osteoblasts in the marrow by
up-regulating the production and action of cytokines that are
responsible for osteoclastogenesis and osteoblastogenesis
(21, 161, 162). Indeed, both estrogen and androgen suppress
the production of IL-6, as well as the expression of the two
subunits of the IL-6 receptor, IL-6R␣ and gp130, in cells of the
bone marrow stromal/osteoblastic lineage (40, 163). Suppression of IL-6 production by estrogen or selective estrogen
receptor modulators (SERMs), such as raloxifene, does not
require direct binding of the estrogen receptor to DNA. Instead, it is due to protein-protein interaction between the
estrogen receptor and transcription factors such as NF-K␤
and C/EBP. This mechanism provides a model that best fits
current understanding of the molecular pharmacology of
estrogen and SERMs (164). Consistent with the suppressive
effect of sex steroids on IL-6 and its receptor, several, albeit
not all, studies have shown that the level of expression of
IL-6, as well as IL-6R␣ and gp130, is elevated in estrogen-
TABLE 2. Cellular changes and their culprits in the three most common causes of bone loss
Sex steroid deficiency
Senescence
Glucocorticoid excess
a
Cellular changes
Probable culprits
1Osteoblastogenesis
1Osteoclastogenesisa
1Lifespan of osteoclasts
2Lifespan of osteoblasts
2Lifespan of osteocytes
2Osteoblastogenesisb
2Osteoclastogenesis
1Adipogenesis
2Lifespan of osteocytes
2Osteoblastogenesis
2Osteoclastogenesis
1Adipogenesis
1Lifespan of osteoclastsc
2Lifespan of osteoblasts
2Lifespan of osteocytes
Increased IL-6; TNF; IL-1RI/IL-RII
MCSF; decreased TGF␤; OPG
Loss of pro- and antiapoptotic effects of sex steroids, respectively
Increased PPAR␥2, PGJ2, noggin; Deceased IL-11, IGFs
Decreased Cbfal and TGF␤ R1; and BMP-2 and IGF1 action
Increased PPAR␥2
Decreased Bcl-2/BAX ratio
Oversupply of osteoclasts relative to the need for remodeling.
Undersupply of osteoblasts relative to the need for cavity repair.
c
Osteoclast numbers may transiently increase in the earlier stages of steroid therapy, without an increase in osteoclastogenesis, indicating
increased lifespan.
b
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MANOLAGAS
deficient mice and rats as well as in humans, in the bone
marrow and in the peripheral blood (165–168). Furthermore,
neutralization of IL-6 with antibodies or knockout of the IL-6
gene in mice prevents the expected cellular changes in the
marrow and in trabecular bone sections and protects the mice
from bone loss after loss of sex steroids (51, 67). Consistent
with its pathogenetic role in the bone loss caused by loss of
sex steroids, IL-6 seems to play a similar role in several other
conditions associated with increased bone resorption as evidenced by increased local or systemic production of IL-6 and
the IL-6 receptor in patients with multiple myeloma, Paget’s
disease, rheumatoid arthritis, Gorham-Stout or disappearing
bone disease, hyperthyroidism, primary and secondary hyperparathyroidism, as well as McCune Albright Syndrome
(66, 68, 169 –174).
In line with the fact that loss of sex steroids increases the
rate of bone remodeling, in addition to up-regulating osteoclastogenesis, loss of sex steroids increases the number of
osteoblast progenitors in the murine bone marrow. These
changes are temporally associated with increased bone formation and parallel the increased osteoclastogenesis and
bone resorption (175). As IL-6 type cytokines can stimulate
osteoblast development and differentiation (54, 55, 146), increased sensitivity to IL-6 and other members of this cytokine’s family may account also for the increased osteoblast
formation that follows the loss of gonadal function. In view
of the fact that mesenchymal cell differentiation and osteoclastogenesis are tightly linked, stimulation of mesenchymal cell differentiation toward the osteoblastic lineage after
sex steroid loss may be the first event that ensues after the
hormonal change, and increased osteoclastogenesis and
bone loss might be downstream consequences of this change
(106).
In addition to IL-6, estrogen also suppress TNF and M-CSF
(176, 177), and estrogen loss may increase the sensitivity of
osteoclasts to IL-1 by increasing the ratio of the IL-1RI over
the IL-1 receptor antagonist (IL-RII) (178). As in the case of
IL-6, the effects of estrogen on TNF and M-CSF are mediated
via protein-protein interactions between the estrogen receptor and other transcription factors. In agreement with the
evidence that IL-1 and TNF play a role in the bone loss caused
by loss of estrogen, administration of IL-1RA and/or a TNF
soluble receptor ameliorates the bone loss caused by ovariectomy in rats and mice (179 –181). Because of the interdependent nature of the production of IL-1, IL-6, and TNF, a
significant increase in one of them may amplify, in a cascade
fashion, the effect of the others (161). Interestingly, recent in
vitro studies with human osteoblastic cells indicate that OPG
production is stimulated by estrogen, suggesting that this
cytokine may also play an important role in the antiosteoclastogenic (antiresorptive) action of estrogen on bone (182).
Increased remodeling, resulting from up-regulation of osteoblastogenesis and osteoclastogenesis, alone can cause a
transient acceleration of bone mineral loss because bone resorption is faster than bone formation, and new bone is less
dense than older bone. However, in addition to increased
bone remodeling, loss of sex steroids leads to a qualitative
abnormality: osteoclasts erode deeper than normal cavities
(133, 183). In this manner, sex steroid deficiency leads to the
removal of some cancellous elements entirely; the remainder
Vol. 21, No. 2
are more widely separated and less well connected. An
equivalent amount of cancellous bone distributed as widely
separated, disconnected, thick trabeculae is biomechanically
less competent than when arranged as more numerous, connected, thin trabeculae. Concurrent loss of cortical bone occurs by enlargement and coalescence of subendocortical
spaces, a process due to deeper penetration of endocortical
osteoclasts.
This deeper erosion can be now explained by evidence that
estrogen acts on mature osteoclasts to promote their apoptosis; consequently, loss of estrogen leads to prolongation
of the lifespan of osteoclasts (133). Specifically, estrogen promotes osteoclast apoptosis in vitro and in vivo by 2- to 3-fold,
an effect seemingly mediated by TGF␤ (139). In direct contrast to their proapoptotic effects on osteoclasts, estrogen (as
well as androgen) exerts antiapoptotic effects on osteoblasts
and osteocytes; consequently, loss of estrogen or androgen
leads to shorter lifespan of osteoblasts and osteocytes (184).
Extension of the working life of the bone-resorbing cells and
simultaneous shortening of the working life of the boneforming cells, can explain the imbalance between bone resorption and formation that ensues after loss of sex steroids.
Furthermore, the increase in osteocyte apoptosis could further weaken the skeleton by impairment of the osteocytecanalicular mechanosensory network. The increase in bone
remodeling that occurs with estrogen deficiency would
partly replace some of the nonviable osteocytes in cancellous
bone, but cortical apoptotic osteocytes might accumulate
because of their anatomic isolation from scavenger cells and
the need for extensive degradation to small molecules to
dispose of the osteocytes through the narrow canaliculi.
Hence, the accumulation of apoptotic osteocytes caused by
loss of estrogen, or glucocorticoid excess (130), could increase
bone fragility even before significant loss of bone mass, because of the impaired detection of microdamage and repair
of substandard bone.
In conclusion, the increased rate of bone remodeling in
estrogen deficiency may be due to increased production of
both osteoclasts and osteoblasts, and the imbalance between
bone resorption and formation is due to an extension of the
working lifespan of the osteoclast and shortening of the
working lifespan of the osteoblast. Moreover, a delay of
osteoclast apoptosis seems responsible for the deeper resorption cavities and thereby the trabecular perforation associated with estrogen deficiency.
Clinical observations of decreased bone mass in a male
with mutant estrogen receptor (185), and increased bone
mass after treatment with estrogen in two males with P-450
aromatase deficiency (186, 187), have raised the possibility
that estrogen derived by peripheral aromatization of androgens is critical for the maintenance of bone mass in men as
well as in women (188). However, in all three cases, the
decreased bone mass in young males with estrogen deficiency in the face of androgen sufficiency could be due to
failure in achieving peak bone mass from defects occurring
during development or growth, not to loss of bone mass, as
it is the case with common forms of osteoporosis. In addition,
individuals with complete androgen insensitivity, due to
mutations in the androgen receptor gene on the X chromosome and increased testosterone and estrogen production,
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BIRTH AND DEATH OF BONE CELLS
have decreased bone mass, in spite of the elevated estrogen
levels (189). Moreover, androgens, including nonaromatizable ones, have identical effects to those of estrogen on the
biosynthetic activity and the birth as well as the death of bone
cells in vitro and in vivo, at least in rodents (67, 106, 190). It
is therefore more likely that both estrogen and androgen are
important for the maintenance of bone mass in the adult male
skeleton.
B. Senescence
The amount of bone formed during each remodeling cycle
decreases with age in both sexes. This is indicated by a
consistent histological feature of the osteopenia that occurs
during aging, namely a decrease in wall thickness, especially
in trabecular bone (191–193). Wall thickness is a measure of
the amount of bone formed in a remodeling packet of cells
and is determined by the number or activity of osteoblasts at
the remodeling site.
Studies measuring bone turnover by histomorphometry
(194), or indirectly by circulating markers (195–197), have
suggested that in aging women, even in extreme old age,
bone turnover is most likely increased by secondary hyperparathyroidism or by the continuing effect of estrogen deficiency. Increased turnover and reduced wall thickness are
not inconsistent, as the former is the result of increased
activation frequency, and the decreased wall thickness—an
index of decreased bone formation by osteoblasts—in senescence is local and relative to the demand created by resorption.
Changes in the birth of bone cells in the bone marrow
provide a mechanistic explanation for the contribution of
senescence to bone loss, independently from sex steroid deficiency. Specifically, using SAMP6 mice, a murine model of
age-related osteopenia (but sufficient in sex steroids and with
intact reproductive function), a tight association among reduced number of osteoblast progenitors, decreased bone formation, and decreased bone mass has been established (105).
Decreased osteoblastogenesis with advancing age has been
confirmed in the human bone marrow (198, 199). Importantly, the decrease in osteoblastogenesis is accompanied by
increased adipogenesis and myelopoiesis, as well as decreased osteoclastogenesis, the latter most likely caused by a
reduction in the stromal/osteoblastic cells that support osteoclast formation (105, 200). This suggests that in aging there
must be changes in the expression of genes that favor the
differentiation of multipotent mesenchymal stem cells toward adipocytes at the expense of osteoblasts. The evidence
that PPAR␥2 induces the terminal differentiation of marrow
cells with both osteoblastic and adipocytic characteristics to
adipocytes, and simultaneously suppresses Cbfa1 expression and terminal differentiation to osteoblasts (94), raises the
possibility that increased expression of PPAR␥2 or its ligands, e.g., PGJ2, may be some of the culprits responsible for
the reciprocal change between adipogenesis and osteoblastogenesis with advancing age (Table 2).
Uptake of oxidized low-density lipoproteins (LDL) play an
important role in foam cell formation and the pathogenesis
of atherosclerosis. Two of the major components of oxidized
LDL, 9-hydroxy-9,11-octadecadienoic acid (HODE) and 13-
127
HODE, are endogenous ligands and activators of PPAR␥
(201), and PPAR␥ promotes monocyte/macrophage differentiation and uptake of oxidized LDL (202). Taken together
with these findings, the evidence that activated PPAR␥2
promotes adipocyte differentiation at the expense of osteoblastogenesis in the bone marrow by suppressing Cbfa1 (94)
suggests a mechanistic link among dietary fat/lipoproteins,
bone marrow stromal cell differentiation, osteoporosis, and
atherogenesis. In support of the existence of such a link,
activation of PPAR␥ by thiazolidinediones or oxidatively
modified LDL inhibits osteoblastogenesis of bone marrowderived stromal cells in vitro (94, 203, 204). Moreover, high
fatty acid content in rabbit serum or high fat diet of mice for
4 months decreases osteogenic cell differentiation in ex vivo
bone marrow cell cultures (86, 204). These new advances may
explain clinical observations that atherosclerosis and osteoporosis coexist (205, 206).
Quantitative trait loci (QTLs) analysis of osteopenia-associated loci using closely related mouse strains have mapped
five loci to regions of chromosomes 2, 7, 11, and 16 (207).
Association of these same loci with bone mineral density has
been reproduced in crosses of different recombinant-inbred
mouse strains (208, 209). Such recurrent appearance of QTL,
especially in crosses involving distantly related strains, implies that polymorphism at these loci may be favored by
evolution and might underlie variation in peak bone density
among humans. Intriguingly, of the more than 12 genes
affecting bone homeostasis that were localized near these
QTLs, 2 are prostaglandin synthases, a third is the BMP-2/4
antagonist noggin, a fourth is the proapoptotic protein bax,
and the fifth is IL-11. Hence, the transcription factor PPAR␥2
and its ligand, PGJ2, noggin, and IL-11 are potentially responsible for the decreased osteoblastogenesis with advancing age. This contention is supported by the evidence that the
reciprocal relationship between decreased osteoblastogenesis and increased adipogenesis in the SAMP6 mouse may be
explained by a change in the expression of PPAR␥ or its
ligands in early mesenchymal progenitors; that BMP-2/4, in
balance with noggin, may determine the tonic baseline control of the rate of osteoblastogenesis; and that IL-11 is a potent
inhibitor of adipogenesis, which stimulates osteoblast differentiation and the expression of which is reduced in
SAMP6 mice. In addition to these factors, growth factors such
as IGFs have also been implicated in the bone loss associated
with senescence (210, 211). Irrespective of the identity of the
precise mediator, the reciprocal change between adipogenesis and osteoblastogenesis can explain the association of
decreased relative bone formation and the resulting osteopenia with the increased adiposity of the marrow seen with
advancing age in animals and humans (105, 142, 212–215).
C. Glucocorticoid excess
The cardinal histological features of glucocorticoid-induced
osteoporosis are decreased bone formation rate, decreased wall
thickness of trabeculae (a strong indication of decreased work
output by osteoblasts), and in situ death of portions of bone.
Increased bone resorption, decreased osteoblast proliferation
and biosynthetic activity, and sex-steroid deficiency, as well as
hyperparathyroidism resulting from decreased intestinal cal-
128
MANOLAGAS
cium absorption and hypercalciuria due to defective vitamin D
metabolism, have all been proposed as mechanisms for the loss
of bone that ensues with glucocorticoid excess (216).
The decreased bone formation and osteonecrosis can now
be explained by evidence that glucocorticoid excess has a
suppressive effect on osteoblastogenesis in the bone marrow
and also promotes the apoptosis of osteoblasts and osteocytes (118). Indeed, mice receiving glucocorticoids for 4
weeks, a period equivalent to ⬃3– 4 yr in humans, exhibit
decreased bone mineral density associated with a decrease in
the number of osteoblast, as well as osteoclast, progenitors in
the bone marrow and a dramatic reduction in cancellous
bone area and in trabecular width compared with placebo
controls. These changes are associated with a significant reduction in osteoid area and a decrease in the rates of mineral
apposition and bone formation. More strikingly, glucocorticoid administration to mice causes a 3-fold increase in the
prevalence of osteoblast apoptosis in vertebrae and induced
apoptosis in 28% of the osteocytes in metaphyseal cortical
bone. Nevertheless, even though there is a significant correlation between the severity of the bone loss and the extent
of reduction in bone formation, some of the bone loss may
be due to an early increase in bone resorption as evidenced
by an early increase in osteoclast perimeter of vertebral cancellous bone after 7 days of steroid treatment. In vivo studies
with mice from this author’s group show that at this early
time point (7 days after glucocorticoid administration) osteoclastogenesis in ex vivo bone marrow cultures is decreased
by half, while the number of osteoclasts in bone sections
doubles (Robert Weinstein, personal communication), suggesting that an early effect of glucocorticoid excess might be
increased osteoclast survival. In vitro studies by others, on the
other hand, have shown that glucocorticoids inhibit OPG and
concurrently stimulate the expression of RANK-ligand in
human osteoblastic, primary, and immortalized bone marrow stromal cells (217). Taken together, these lines of evidence suggest that the initial rapid phase of bone loss with
glucocorticoid treatment could be due to an extension of the
lifespan of preexisting osteoclasts, mediated by RANKligand (117).
The same histomorphometric changes that have been
found in mice after a 4-week treatment with steroids have
been confirmed in biopsies from patients receiving long-term
glucocorticoid therapy. Moreover, as in mice, an increase in
osteoblast and osteocyte apoptosis is found in human biopsies. Compared with osteoblast apoptosis, apoptotic osteocytes are far more prevalent, at least in metaphyseal cortices,
probably because of the anatomical isolation of osteocytes
from scavenger cells. Consistent with these findings, glucocorticoids promote osteoblast and osteocyte apoptosis in
vitro (218, 219). Decreased production of osteoclasts can explain the reduction in bone turnover with chronic glucocorticoid excess, whereas decreased production and apoptosis
of osteoblasts can explain the decline in bone formation and
trabecular width. Accumulation of apoptotic osteocytes may
also explain the so-called “osteonecrosis,” also known as
aseptic or avascular necrosis, another manifestation of steroid-induced osteoporosis that causes collapse of the femoral
head in as many as 25% of patients (220). This contention is
supported by evidence that whole femoral heads obtained
Vol. 21, No. 2
from patients with glucocorticoid-induced osteoporosis exhibit abundant apoptotic osteocytes adjacent to the subchondral fracture crescent (221). Glucocorticoid-induced osteocyte apoptosis, a cumulative and unrepairable defect, could
uniquely disrupt the proposed mechanosensory role of the
osteocyte network and thus promote collapse of the femoral
head.
At this time, the mediators of the cellular changes caused
by glucocorticoid excess are only a matter of conjecture.
Nonetheless, glucocorticoids directly suppress BMP-2 and
Cbfa1-2—two critical factors for osteoblastogenesis—and
may also decrease the production of IGFs while they stimulate the transcriptional activity of PPAR␥2 (222–225) (Table
2).
X. Pharmacotherapeutic Implications of Osteoblast
and Osteocyte Apoptosis
Estrogen replacement therapy (ERT), various bisphosphonates (e.g., alendronate), the SERM raloxifene, calcitonin, and
sodium fluoride, as well as calcium and vitamin D, are approved modalities for the prevention and treatment of bone
loss, irrespective of its cause. Decreased osteoclast progenitor
development and/or decreased osteoclast recruitment and
promotion of apoptosis of mature osteoclasts leading to a
slowing of the rate of bone remodeling are thought to be the
main mechanisms of the so-called “antiresorptive” agents
estrogen, bisphosphonates, SERMs, and calcitonin. Sodium
fluoride has anabolic properties, but its therapeutic range is
very narrow. Calcium and vitamin D are rarely sufficient on
their own, but they are considered a very useful supplementation in any regimen for osteoporosis.
A. Intermittent PTH administration
The ideal therapy for osteoporosis, especially in elderly
patients who already have advanced bone loss, would be an
anabolic agent that will increase bone mass by rebuilding
bone. It is well established that daily injections of low doses
of PTH—an agent better known for its role in calcium homeostasis—increases bone mass in animals and humans
(226 –231) as does the PTH-related protein (PTHrP), the only
other known ligand of the PTH receptor (232). Indeed, although constant, high levels of PTH cause increased bone
resorption and osteitis fibrosa cystica, low and intermittent
doses of PTH, too small to affect serum calcium concentrations, promote bone formation and increase bone mineral
density at the lumbar spine and hip. This so-called anabolic
effect can be now explained by evidence that PTH increases
the life span of mature osteoblasts in vivo by reducing the
prevalence of their apoptosis from 1.7–2.2% to as little as
0.1– 0.4% rather than by affecting the generation of new osteoblasts (218). The antiapoptotic effect of PTH in mice was
sufficient to account for the increase in bone mass and was
confirmed in vitro using rodent and human osteoblasts and
osteocytes. Like PTH, PGE inhibits periosteal cell apoptosis
via cAMP-dependent stimulation of sphingosine kinase
(233). Interestingly, whereas PTH inhibits apoptosis in cells
overexpressing Gs, an activator of adenylate cyclase, PTH
stimulates apoptosis via G protein-coupled receptors in cells
April, 2000
BIRTH AND DEATH OF BONE CELLS
overexpressing Gq (an activator of JNK and calcium signaling), suggesting that the antiapoptotic effects of PTH are
mediated by signals transduced through the Gs pathway
(234).
Osteocytes in the newly made lamellar cancellous bone
in the mice receiving daily PTH injections were closer
together and more numerous than those found in the
animals receiving vehicle alone (218). The closely spaced,
more numerous osteocytes are the predictable consequence of protecting osteoblasts from apoptosis (Fig. 3B).
The antiapoptotic effect of PTH on osteoblasts as well as
osteocytes has been confirmed in vitro using primary bone
cell cultures and established cell lines. Elucidation of this
mechanism provides for the first time proof that inhibition
of osteoblast apoptosis may represent a novel therapeutic
strategy for augmenting bone mass. Be that as it may,
several alternative mechanisms, including activation of
lining cells, have been proposed, and they may also contribute to the anabolic effect of PTH (235–237). Nonetheless, lining cells cover at least 3 times more surface than
osteoblasts. Therefore, conversion of lining cells to boneforming osteoblasts alone would be insufficient to cover
the increased cancellous bone area observed in rats and to
account for the expanded bone perimeter and the increased osteocyte number and density observed with PTH
treatment in mice (218).
Daily subcutaneous injections of PTH are safe and effective in the treatment of patients with corticosteroid-induced
osteoporosis (230). The elucidation of the importance of osteoblast and osteocyte apoptosis in the mechanism of glucocorticoid-induced osteoporosis, and the elucidation of the
importance of preventing apoptosis in the anabolic effects of
PTH on bone, readily explain how PTH can be such an
effective therapy in this condition. Hence, PTH and perhaps
future PTH mimetics represent, for the first time, pathophysiology-based, i.e., rational as opposed to empirical, pharmacotherapies for osteopenias, in particular, those in which
osteoblast progenitor formation is suppressed. In any case,
future studies to assess the antifracture efficacy of these
compounds will be needed before their effectiveness for the
management of osteoporosis can be established.
B. Bisphosphonates and calcitonin
Bisphosphonates, stable analogs of pyrophosphate, and
calcitonin are potent inhibitors of bone resorption and effective therapies for the management of osteoporosis and
other diseases characterized by bone loss (238, 239). The main
mechanism of the antiresorptive actions of these agents is
decreased development of osteoclast progenitors, decreased
osteoclast recruitment, and promotion of apoptosis of mature
osteoclasts leading to a slowing rate of bone remodeling (133,
240 –242). Nonetheless, the antifracture efficacy of these
agents is disproportional to their effect on bone mass (243),
suggesting an additional effect on bone strength unrelated to
effects on bone resorption. Moreover, long-term treatment of
human and nonhuman primates with bisphosphonates has
been shown to increase wall thickness, an index of increased
osteoblast numbers or activity (244 –246), raising the possibility that they may not only inhibit bone resorption, but may
129
also have a positive effect on bone formation. An explanation
for this evidence is now provided by studies demonstrating
that bisphosphonates such as etidronate, alendronate, pamidronate, olpadronate, or amino-olpadronate (IG9402, a
bisphosphonate that lacks antiresorptive activity), as well as
calcitonin have antiapoptotic effects on osteoblasts and osteocytes (219). These effects are associated with a rapid increase in the phosphorylated fraction of extracellular regulated signal kinases (ERKs) and are blocked by specific
inhibitors of ERK activation. In agreement with the in vitro
results, alendronate abolishes the increase in the prevalence
of vertebral, cancellous bone osteocyte and osteoblasts apoptosis induced by administration of prednisolone in mice.
These findings raise for the first time the possibility that
increased survival of osteoblasts and osteocytes may both
contribute to the efficacy of bisphosphonates and calcitonin
in the management of disease states due to loss of bone.
If both “antiresorptive” and “anabolic” agents [e.g., intermittent PTH] prevent osteoblast and osteocyte apoptosis, why is increased formation so much more apparent in
the case of the “anabolic” agents? The discussion of physiological bone regeneration in the beginning of this review
article provides an answer. Bone formation occurs only on
sites of previous osteoclastic bone resorption, i.e., on sites
undergoing remodeling. Each remodeling cycle is a transaction that, once consummated, is irrevocable. Therefore,
agents with antiapoptotic properties that do not have antiresorptive/antiremodeling properties, i.e., they do not
decrease the number of remodeling units, are expected to
rebuild more bone and therefore increase the overall bone
mass, because of the greater number of profitable transactions. Hence, by decreasing the prevalence of osteoblast
apoptosis, agents with pure antiapoptotic properties, such
as intermittent PTH, can expand the pool of mature osteoblasts at sites of new bone formation and allow these
cells more time to make bone, to a much greater degree
than the antiresorptive agents that also slow remodeling.
However, in the case of either class of agents, upholding
the osteocyte-canalicular network by preventing osteocyte
apoptosis, should contribute to antifracture efficacy, over
and above that resulting from their effects on bone mass
(Fig. 5). Therefore, the distinction between antiresorptive
and anabolic agents may be more apparent than real when
it comes to antifracture efficacy.
C. Novel pharmacotherapeutic strategies
Based on the understanding of the role of growth factors
on osteoblast development, proliferation, and differentiation, several of them (e.g., GH, the insulin-like growth factors
I and II, TGF␤, BMPs, and FGF) have been advocated as
potential future therapeutic agents for the management of
bone loss (247, 248). However, with the exception of BMPs,
which may be of value in local augmentation of bone mass
and acceleration of fracture healing, none of them has been
shown to be efficacious (let alone safe or convenient and
practical) for the management of common metabolic bone
disorders such as osteoporosis. The recent elucidation of the
mechanism of the anabolic effects of PTH, and specifically
the demonstration of increased work output of a cell pop-
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MANOLAGAS
Vol. 21, No. 2
FIG. 5. Hypothetical model of the implications of the effects of antiresorptive (i.e., antiremodeling) vs. non-antiresorptive agents on prolonging the life
span of mature osteoblasts and osteocytes. For explanation, please see text.
ulation by suppressing apoptosis to augment tissue mass,
points to an entirely new avenue for future drug discovery.
Indeed, in addition to PTH and PTH mimetics, which by
virtue of their peptidic nature carry the inconvenience of
daily injections, one can for the first time envisage nonpeptide inhibitors of proapoptotic pathways in osteoblasts and
osteocytes as therapeutic agents for osteopenias and especially those in which osteoblast progenitors are low, such as
age-related and glucocorticoid-induced osteoporosis.
As discussed in Section VIII in this review, estrogen and
androgen deficiency increase osteoblast and osteocyte apoptosis in humans, rats, and mice; and these changes have
been shown to be reversed by replacement therapy, at least
in mice (132, 144, 184, 190). In full agreement with these in
vivo observations, 17␤-estradiol inhibits osteoblast and osteocyte apoptosis in vitro. The antiapoptotic effect of 17␤estradiol on osteoblasts and osteocytes require the presence
of the estrogen receptor-␣ or -␤ (249). Nonetheless, unlike the
classical mechanism of estrogen receptor action that involves
direct or indirect interaction with the transcriptional apparatus, the estrogen receptor-dependent antiapoptotic effect
of 17␤-estradiol is due to rapid (within 5 min) phosphorylation of ERKs (250). Moreover, the antiapoptotic effect of
17␤-estradiol can be reproduced by 17␣-estradiol, a compound thought of as an inactive analog of 17␤-estradiol, as
well as a membrane-impermeable conjugate of 17␤-estradiol
with BSA (17␤E2-BSA). Numerous effects of estrogen have
been observed over the last few years in a variety of cell
types, including osteoblasts, the rapidity of which makes a
genomic mechanism of action unlikely (251–256). Many of
these rapid actions have been attributed to the ability of
estrogen to act at the cell membrane on a membrane-associated estrogen receptor (257–260). The antiapoptotic effects
of estrogen on osteoblasts and osteocytes fall into this category of “nongenomic” actions. Based on this, the term “activators of non-genomic estrogen-like signaling” (ANGELS),
has been coined for compounds that mimic the nongenomic
effects of estrogen, but have reduced classical estrogenic
actions (261). A paradigm of such agents is the synthetic
compound estratriene-3-ol, which has decreased transcriptional activity as compared with 17␤-estradiol (262, 263), is
a potent neuroprotective compound (264 –266), and does exhibit potent antiapoptotic effects on osteoblasts and osteocytes in vitro. In support of the hypothesis that ANGELS can
be used as a novel, advantageous mode of therapy for the
augmentation of bone mass and/or fracture prevention in
diseases characterized by low bone mass and increased fragility, preliminary evidence indicates that estratriene-3-ol
increases BMD and bone strength in both estrogen-replete
and estrogen-deficient mice (261). In view of this preclinical
finding and the evidence that androgen (190), as well as
estrogen, have antiapoptotic effects on osteoblasts and osteocytes, one is encouraged to think that estrogenic, androgenic, or even nonsteroidal compounds that can activate
antiapoptotic, but not antiremodeling, signals on osteoblasts
and osteocytes, are candidates for future osteoporosis treatments that, unlike existing ones that prevent or retard bone
loss, may augment bone mass.
XI. Summary and Conclusions
In 1995, it was proposed that “changes in the numbers of
bone cells, rather than changes in the activity of individual
cells, form the pathogenetic basis of osteoporosis”; and that
“excessive osteoclastogenesis and inadequate osteoblastogenesis are responsible for the mismatch between the formation and resorption of bone in postmenopausal and agerelated osteopenia” (21). Since then, this paradigm shift of
thinking has led to important new discoveries that, along
with several other independent breakthroughs, refine the
concept of “cell number” and broaden its relevance to the
physiology and pathophysiology of bone at large. Moreover,
these discoveries provide a new landscape for critical reevaluation of our current thinking about therapeutic strategies for bone diseases. Indeed, it is now clear that bone cells
must be continually replaced, and the number present depends not only on their birth rate, which reflects the frequency of cell division of the appropriate precursor cell, but
also on the life span, which most likely reflects the timing of
death by apoptosis. The process of replacement of osteoblasts
and osteoclasts is tightly coordinated and orchestrated at the
April, 2000
BIRTH AND DEATH OF BONE CELLS
early progenitor level. Changes in the birth rate and/or apoptosis of bone cells may account for previously unexplained
bone diseases, such as the osteoporosis caused by sex steroid
deficiency, old age, and glucocorticoid excess. Moreover,
attenuation of the rate of apoptosis of osteoblastic cells may
be a key mechanism for the effects of anabolic agents, such
as PTH. Proof of the principle that the work performed by a
cell population can be increased by suppression of apoptosis
provides clues for the development of novel pharmacotherapeutic strategies for pathological conditions such as osteoporosis in which tissue mass diminution has compromised
functional integrity. Nevertheless, changes in cell birth and
death, as well as other mechanisms including changes in
bone cell activity, need to be investigated in humans more
extensively before definitive conclusions on the pathogenesis
of the various causes of bone loss and the development of
osteoporosis can be reached.
Acknowledgments
The author is grateful to A. Michael Parfitt, Robert L. Jilka, Robert S.
Weinstein, Teresita Bellido, Charles A. O’Brien, Robert S. Reis, Paula
Roberson, Beata Lecka-Czernik, Etsuko Abe, Donald L. Bodenner, and
Stavroula Kousteni for many insightful discussions, sharing of ideas,
and contribution of research findings during the preparation of the
manuscript; and to Tonya Smith for secretarial assistance.
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STADY II-International Symposium on Signal Transduction in Health and Disease
September 12–15, 2000
Tel Aviv, Israel
For further information, contact: Professor Zvi Naor
Department of Biochemistry
Tel Aviv University
Tel Aviv, Israel
Telephone: 972-3-640-9032/641-7057
Fax: 972-3-640-6834
E-mail: [email protected] or [email protected]
137
IBMS BoneKEy. 2011 February;8(2):74-83
http://www.bonekey-ibms.org/cgi/content/full/ibmske;8/2/74
doi: 10.1138/20110493
PERSPECTIVES
The Osteoclast Cytoskeleton: How Does It Work?
Steven L. Teitelbaum and Wei Zou
Washington University School of Medicine, St. Louis, Missouri, USA
Abstract
The capacity of the osteoclast to resorb bone is distinctive, as is the cell’s appearance. Both
characteristics reflect cytoskeletal organization that yields structures such as the sealing zone and ruffled
border. The unique nature of these organelles and their dependence upon contact with bone have been
appreciated for some time but insights into the mechanisms by which they are generated come from more
recent studies. These insights include the role of integrins, particularly αvβ3, in cytoskeletal organization and
the canonical signaling pathway they activate. Investigators now appreciate that the sealing zone isolates
the resorptive microenvironment from the general extracellular space, permitting secretion of matrixdegrading molecules on the bone surface. Thus, the osteoclast is a secretory cell that depends upon
polarization of exocytic vesicles to the bone-apposed plasma membrane into which they insert under the
aegis of vesicle/membrane fusion proteins. This process focally expands and convolutes the plasmalemma
included within the sealing zone, eventuating in formation of the ruffled border. Many of these events are
now better understood and are the focus of this Perspective. IBMS BoneKEy. 2011 February;8(2):74-83.
2011 International Bone & Mineral Society
Isolating
the
Microenvironment
Resorptive
Osteoclasts are polykaryons and members
of the macrophage lineage with the unique
capacity to degrade the inorganic and
organic matrices of bone. If excessive, the
bone resorptive activity of osteoclasts
causes osteoporosis. Conversely, the
osteoclast initiates remodeling that likely
removes structurally compromised bone,
thereby maintaining mechanical integrity (1).
Skeletal health, therefore, requires optimal
osteoclast function.
Resorption is initiated by attachment of
osteoclasts to bone. They then develop a
compartment
between
their
plasma
membrane and the bone surface into which
the
cells
transport
matrix-degrading
+
molecules including H and Cl , which, in
concert, demineralize the target bone, and
cathepsin K, which degrades the exposed
collagen fibers and associated proteins. To
isolate this resorptive microenvironment
from the general extracellular space,
osteoclasts reorganize their cytoskeleton to
generate an encompassing, actin-rich,
gasket-like, sealing zone.
A single
osteoclast, being a large cell, enjoys multiple
contacts with bone and therefore generates
numerous sealing zones and resorptive
microenvironments.
When most other cells attach to matrix they
generate focal adhesions. These stable
structures contain integrins and signaling
and cytoskeletal molecules that, upon
contact with matrix, promote formation of
actin stress fibers. Consistent with the lack
of actin stress fibers in mammalian
osteoclasts, they lack focal adhesions but in
their place develop podosomes (2;3). These
punctuate structures contain an F-actin core
and a peripheral “cloud” of a loose network
of radial actin cables (4-7). In contrast to
focal adhesions, podosomes are transient
but mediate substrate adhesion and thus
formation
of
the
resorptive
microenvironment.
Most past studies of osteoclast podosomes
utilized cells resident on plastic or glass. In
these
circumstances,
the
punctuate
structures initially appear in clusters but
ultimately
coalesce,
first
into
an
74
Copyright 2011 International Bone & Mineral Society
IBMS BoneKEy. 2011 February;8(2):74-83
http://www.bonekey-ibms.org/cgi/content/full/ibmske;8/2/74
doi: 10.1138/20110493
intracytoplasmic actin ring and then a
peripheral actin belt (4). The physiological
relevance
of
this
observation
was
challenged by the absence of apparent
podosomes or an actin belt in non-stressed
osteoclasts on bone (4). While the relevance
of the peripheral belt is not established, it is
clear that, like the actin ring, the sealing
zone of bone-residing osteoclasts also
consists of podosome-containing structural
units (8).
Microtubules Are Important
Depending upon their state of acetylation,
microtubules in osteoclasts are transient or
polymerized and relatively stable (9-11).
Unlike actin ring formation by glass-residing
cells, sealing zone generation in boneresorbing osteoclasts is characterized by
microtubule acetylation, again illustrating the
influence of substrate on the cell’s
cytoskeleton (10).
The
histone
deacetylase
HDAC6
depolymerizes tubulin, thereby destabilizing
microtubules. Cbl proteins compete with the
deacetylase for tubulin binding and thus
promote
polymerization
(12).
While
destabilizing microtubules in other cells (13),
RhoA appears to activate HDAC6 in glassgenerated osteoclasts, suggesting the
GTPase negatively regulates the cell. When
on bone, however, osteoclasts in which
RhoA is inhibited lose their apical-basal
polarity and, consequently, are incapable of
optimal resorption (6). Furthermore, RhoA
promotes actin ring and podosome
formation and osteoclast motility (6;14).
When osteoclasts contact bone, RhoA is
activated and localizes to the cytoskeleton
(15;16), The fact that RhoA activation is
diminished, in osteoclasts lacking αvβ3,
establishes that the GTPase is regulated by
the integrin (3). Because of the conflicting
phenotypes of osteoclasts on plastic or
bone, the impact of active RhoA on stability
of their microtubules remains unknown.
degrading bone and its absence indicates
the cell is not doing so. Reflecting multiple
contacts with bone and attendant sealing
zones, ruffled borders are also numerous in
a given osteoclast.
This complex enfolding of the plasma
membrane, unique to the osteoclast, abuts
and extends into the resorptive space. It is
surrounded by the sealing zone and is the
venue by which the cell secretes matrixdegrading molecules on the bone surface
(17). It is therefore the resorptive organelle
and is absent or deranged in many forms of
osteopetrosis. As osteoclast resorption
alternates with migration, the ruffled border
is a transient structure.
The ruffled border, which forms only upon
contact with mineralized substrate, is
initiated by transport of cathepsin K – and/or
+
H ATPase – and Cl channel-bearing
vesicles to the bone-apposed plasma
membrane (17), likely under the aegis of
GTPases such as Rabs 7, 9 and 3 (18-22).
While unproven, studies in chicken
osteoclasts raise the possibility that ruffled
border-forming vesicles may polarize to the
resorptive surface via microtubules (23). The
polarized vesicles fuse with the boneapposed plasma membrane, to increase its
complexity, via a process mirroring
exocytosis (17). Similar to that occurring in
the context of neurotransmitter exocytosis,
vesicle/plasmalemma fusion is regulated by
v- (vesicular) and t- (target) SNAREs
(soluble N-ethylmaleimide-sensitive fusion
protein (NSF) attachment protein (SNAP)
receptors) (24).
The Ruffled Border Is King
Ruffled border formation requires a
synaptotagmin (Syt) linking the vesicle and
target plasma membrane. Fifteen Syt
isoforms have been identified in mammalian
cells but Syt VII generates ruffled borders
and is essential for bone degradation (17).
Syt VII also enables osteoblasts to secrete
bone matrix proteins and as such, both
resorption and formation are repressed in
Syt VII-deficient mice.
The ruffled border is the morphological sine
qua non of the resorbing osteoclast as only
its presence assures that the cell is
Autophagy is a cellular degradative process
by which cells recycle organelles and longlived proteins. Autophagosomes, which are
75
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IBMS BoneKEy. 2011 February;8(2):74-83
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doi: 10.1138/20110493
double-membrane bound vesicles, envelop
and then deliver cellular components to
lysosomes for degradation. While the
process promotes survival in starved or
stressed cells as well as maintenance of
organelle quality (25-27), recent evidence
indicates it may also participate in regulated
exocytosis (28). In fact, Atg5, Atg7 and
LC3β autophagy proteins, representing the
two ubiquitin-like conjugation systems, are
important for generation of the osteoclast
ruffled border and the secretory function of
osteoclasts both in vitro and in vivo. Thus,
osteoclasts lacking these proteins do not
efficiently polarize cathepsin K to the
resorptive microenvironment and are
incapable of optimal bone resorption (29).
Integrin Activity Starts It All
Skeletal resorption requires osteoclast-bone
recognition that is mediated by α/β
heterodimers known as integrins. β1containing heterodimers probably participate
in the process but αv/β3 is the key integrin
regulating skeletal degradation (30). While
the αv subunit is constitutively expressed
throughout
osteoclastogenesis,
the
associated β chains alter with differentiation.
Specifically, immature osteoclast precursors,
in the form of bone marrow macrophages,
express abundant αvβ5 and little αvβ3. As
the cells commit to the osteoclast
phenotype, the magnitude of expression of
the two heterodimers reverses (31). Hence,
αv/β3 is a relatively specific marker of
osteoclastogenesis. This integrin is liganded
by the amino acids Arg-Gly-Asp (RGD), a
motif present in bone proteins such as
osteopontin and bone sialoprotein. Small
molecules
mimicking
this
sequence
suppress osteoclast activity and are
candidate anti-resorptive drugs (32-34).
In keeping with its governance of osteoclast
function, absence of αv/β3, globally and
conditionally in osteoclasts, increases bone
mass and protects against estrogendeficient osteoporosis (35;36). Reflecting the
integrin’s role in cytoskeletal organization,
αv/β3 deficiency yields deranged ruffled
borders, failure of cell spreading and suboptimal bone resorption (35). Consequently,
β3(-/-) mice are hypocalcemic and
osteoclast number is substantially increased
in these animals, likely reflecting secondary
hyperparathyroidism and an abundance of
osteoclastogenic cytokines in the marrow
(35;37;38). However, in contrast, absence of
αv/β3 diminishes the abundance of the
polykaryons in vitro (33). As differentiation,
apoptosis and precursor proliferation are not
compromised, a reasonable hypothesis
holds that the paucity of osteoclasts, in
culture, reflects cytoskeletal dysfunction,
and specifically impairment of migration
necessary for cell fusion.
αv/β3 signaling in osteoclasts is initiated by
changing the integrin’s conformation from a
low to a high affinity state by outside-in or
inside-out activation (3;39). Outside-in
activation is characterized by integrin
clustering, thereby increasing avidity and
affinity. Inside-out activation is an indirect
event wherein signals emanating from
liganded growth factor or cytokine receptors
target the integrin’s intracellular region,
changing its conformation and consequently,
that of the extracellular domain (40). As will
be discussed, the adaptor protein, talin, is
essential for inside-out αv/β3 activation in
osteoclasts and its absence arrests bone
resorption.
Resorption is a cyclical event wherein a
portion of the osteoclast migrates to a
candidate bone resorptive site and forms an
actin ring and ruffled border. Following
matrix degradation, the cell detaches and reinitiates the cycle. Prior to bone recognition,
the integrin is predominantly in a low affinity
state and confined to podosomes within the
sealing zone (3;41). Activated αv/β3 leaves
the podosome and transits to lamellipodia
that mediate motility, compromised in the
absence of the integrin (3). During
resorption, the heterodimer appears in the
ruffled membrane (3;41).
Integrins serve as attachment molecules but
their intracellular transmission of matrixderived signals is at least as important. For
example, αv/β3 substrate robustly activates
ERKs in wild-type osteoclastic cells but not
those lacking the integrin (3). Since
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doi: 10.1138/20110493
activation of these MAP kinases typically
stimulates proliferation, their absence in β3
knockout osteoclasts may contribute to their
reduced numbers in vitro.
How Does αv/β 3 Do It?
In 1991, Soriano et al. determined that c-src
deletion eventuates in severe osteopetrosis
due to osteoclast dysfunction (42).
Interestingly, c-src-deficient mice, like those
lacking αv/β3, have increased numbers of
osteoclasts that fail to organize their
cytoskeleton. c-src is a tyrosine kinase and
an adaptor protein and both functions are
necessary
for
optimal
cytoskeletal
organization (43).
Because
c-srcand
αv/β3-deficient
osteoclasts share qualitatively similar
cytoskeletal features, the kinase presents as
a mediator of integrin signaling. In fact,
under the aegis of phospholipase Cγ
(PLCγ2) (44), c-src binds directly to the β3
subunit in the bone resorptive cell, and we
have found this to be a constitutive event
(45). Others, however, propose that αv/β3
occupancy
phosphorylates
the
focal
adhesion kinase family member, Pyk2,
which recruits c-src to the integrin (44;46).
αv/β3-associated c-src phosphorylates c-Cbl
which, in turn, inhibits c-src’s activity (3;47).
Regardless
of
the
mechanism
of
association, c-src activation requires integrin
occupancy and again, signaling via PLCγ2.
Activated
c-src
prompts
podosomal
disassembly,
most
probably
by
phosphorylating cortactin (48;49). Hence,
podosomes are more abundant in c-src(-/-)
than wild-type osteoclasts and the mutant
cells are less motile. In keeping with Pyk2
regulating
the
cell’s
cytoskeleton,
osteoclasts lacking the kinase are unable to
generate normal sealing zones on bone
(11). The cytoskeletal effects of Pyk2,
however, may reflect its promotion of tubulin
acetylation.
Syk is another non-receptor tyrosine kinase
mediating αv/β3 signaling in osteoclasts.
Upon integrin occupancy it binds the β3
cytoplasmic domain close to c-src, which
activates it (45). Syk is also negatively
regulated by the ubiquitinating activity of cCbl (50;51).
The ITAM-bearing adaptors, Dap 12 and
FcRγ, are expressed by osteoclasts and
their combined, but not individual deletion
prompts severe osteopetrosis (45;52-54).
While deletion of both co-stimulatory
molecules
is
reported
to
arrest
osteoclastogenesis (55), we find such is not
the case (56), suggesting their resorptive
abnormality reflects deranged osteoclast
function but not generation. The same
obtains
regarding
osteoblast-mediated
generation of osteoclasts lacking Dap12,
with or without FcRγ. These mutant cells
form in normal numbers but fail to organize
their cytoskeleton or resorb bone. Among
the most dramatic consequences of this
dysfunction is the inability of Dap12(-/-)
osteoclasts to migrate through a layer of
osteoblasts, required to attach to a
candidate resorptive bone surface (56;57).
Thus the dominant role of ITAM proteins in
the osteoclast appears to be cytoskeletal
organization and not differentiation (56;58).
Syk-mediated organization of the osteoclast
cytoskeleton involves Vav3. This guanine
nucleotide exchange factor (GEF) is
uniquely expressed in abundance in the cell
and activated upon αv/β3 occupancy in a
SLP-76-dependent manner (15;59). Vavs
transit
cytoskeleton-organizing
Rho
GTPases from their inactive GDP- to their
active GTP-associated conformation. Thus,
Vav3(-/-) osteoclasts are dysfunctional and
the mice from which they are derived are
osteopetrotic.
Vavs are Rac GEFs and it is therefore not
surprising that this Rho GTPase regulates
the osteoclast cytoskeleton in an αv/β3dependent manner (60;61). The two
isoforms expressed in osteoclasts, Rac1
and Rac2, are mutually compensatory (62).
Effective deletion of both, however,
produces severe osteopetrosis in which
osteoclasts
fail
to
organize
their
cytoskeleton. Absence of the related Rho
family GTPase, cdc42, also causes
osteopetrosis but in this circumstance the
dominant mechanism is arrested osteoclast
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doi: 10.1138/20110493
Fig. 1. Proposed mechanism organizing the cytoskeleton of resorbing osteoclasts. 1). M-CSF occupying its
receptor, c-fms, stimulates inside-out αvβ3 activation by inducing talin association with the β3 cytoplasmic
domain that binds c-src constitutively. 2). Clustering of the integrin by RGD ligand increases avidity as well
as affinity by outside-in activation. The liganded integrin activates c-src as evidenced by Y416
phosphorylation. Activated c-src tyrosine phosphorylates ITAM proteins that recruit Syk to the integrin by
binding Syk-SH2 domains. c-src activates β3-associated Syk that phosphorylates Vav3 in the context of
SLP-76. Vav3 then shuttles Rac-GDP to its activated GTP-associated state. 3). Rac-GTP prompts
association of lysosome-derived secretory vesicles with microtubules (MTs) that deliver them to the boneapposed plasma membrane into which they insert under the influence of Syt VII and LC3. Rac-GTP and
MTs also promote sealing zone (SZ) formation. Secretory vesicle fusion focally expands the plasma
membrane forming the ruffled border and eventuating in discharge of cathepsin K (CTK) and HCl into the
resorptive microenvironment.
recruitment due to inhibited precursor
proliferation and accelerated apoptosis of
the mature polykaryon (63).
M-CSF Helps αv/β3
RANK ligand (RANKL) and M-CSF are the
requisite osteoclastogenic cytokines but
each also promotes the resorptive activity of
the mature polykaryon. In the case of MCSF, the cytokine interacting with its
receptor, c-fms, stimulates a signaling
pathway remarkably similar to that induced
by αv/β3,
thereby
organizing
the
cytoskeleton (37;58;64). The means by
which M-CSF structures the osteoclast
cytoskeleton may, therefore, be independent
of the integrin, or alternatively, represent
inside-out αv/β3 activation. In fact, M-CSF
transits αv/β3 from its default low affinity to
its high affinity conformation by inducing
talin binding to the β3 cytoplasmic domain
(39). Absence of talin, in osteoclast
precursors, does not arrest differentiation
but blocks substrate adherence and motility.
The impaired function of talin-deficient
osteoclasts results in a 5-fold increase in the
bone mass of mutant mice. Interestingly, the
osteopetrotic phenotype of mice with talin
(-/-) osteoclasts is more severe than of those
78
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doi: 10.1138/20110493
lacking αv/β3, which likely represents arrest
of compensatory integrins, particularly those
bearing β1 (30;35).
Conclusion
The magnitude of bone resorption reflects
osteoclast number and function of the
individual cell, the latter dependent upon
cytoskeletal organization. The osteoclast
cytoskeleton is a unique structure whose
conversion to its active state depends upon
contact with mineralized matrix (Fig. 1).
These extracellular signals, which polarize
the resorptive machinery to the bone-cell
interface, are transmitted intracellularly by
integrins dominated by αv/β3. In conjunction
with M-CSF-stimulated inside-out activation,
a canonical signaling pathway emanates
from the αv/β3 integrin. Occupancy of the
heterodimer phosphorylates constitutively
associated c-src which in turn targets Dap
12. The ITAM’s phosphotyrosines serve to
recruit Syk to the β3 cytoplasmic domain
where it is also phosphorylated by c-src.
Utilizing SLP-76, Syk activates Vav3,
eventuating in formation of Rac-GTP and
organization of the resorptive cytoskeleton.
Given current concerns regarding longacting anti-resorptive agents, such as
bisphosphonates, short-acting counterparts
are in demand. Delineating the molecular
mechanism by which osteoclasts organize
their cytoskeleton to degrade bone has
provided an array of candidate therapeutic
targets.
Conflict of Interest: None reported.
Peer Review: This article has been peer-reviewed.
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83
Copyright 2011 International Bone & Mineral Society
R E V I E W
Osteoclast Activity and Subtypes as a Function of
Physiology and Pathology—Implications for Future
Treatments of Osteoporosis
K. Henriksen, J. Bollerslev, V. Everts, and M. A. Karsdal
Nordic Bioscience A/S (K.H., M.A.K.), DK-2730 Herlev, Denmark; Section of Endocrinology (J.B.), Department of
Medicine, Rikshospitalet, Oslo University Hospital and the University of Oslo, 0450 Oslo, Norway; and Department Oral
Cell Biology (V.E.), Academic Centre of Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam,
Research Institute Move, 1066 EA Amsterdam, The Netherlands
Osteoclasts have traditionally been associated exclusively with catabolic functions that are a prerequisite for
bone resorption. However, emerging data suggest that osteoclasts also carry out functions that are important
for optimal bone formation and bone quality. Moreover, recent findings indicate that osteoclasts have different
subtypes depending on their location, genotype, and possibly in response to drug intervention.
The aim of the current review is to describe the subtypes of osteoclasts in four different settings: 1) physiological,
in relation to turnover of different bone types; 2) pathological, as exemplified by monogenomic disorders;
3) pathological, as identified by different disorders; and 4) in drug-induced situations.
The profiles of these subtypes strongly suggest that these osteoclasts belong to a heterogeneous cell population,
namely, a diverse macrophage-associated cell type with bone catabolic and anabolic functions that are dependent on both local and systemic parameters. Further insight into these osteoclast subtypes may be important
for understanding cell– cell communication in the bone microenvironment, treatment effects, and ultimately
bone quality. (Endocrine Reviews 32: 31– 63, 2011)
I.
II.
III.
IV.
Introduction
Bone Remodeling
The Classical Osteoclast
Osteoclast Subtypes in Physiological Situations
A. Endochondral vs. intramembranous bone
osteoclasts
B. Chondroclasts
C. Osteoclasts involved in targeted and stochastic
remodeling
D. Trabecular and cortical osteoclasts
E. Diurnal variation in osteoclasts or osteoclast
activity?
V. Osteoclast Subtypes in Pathological Situations
A. Osteoporotic osteoclasts
B. Changes in osteoclast activities with increasing
bone matrix age
C. Osteoclast-rich osteopetrosis
D. Osteoclast-poor osteopetrosis
E. Pycnodysostotic osteoclasts
ISSN Print 0021-972X ISSN Online 1945-7197
Printed in U.S.A.
Copyright © 2011 by The Endocrine Society
doi: 10.1210/er.2010-0006 Received March 19, 2010. Accepted August 10, 2010.
First Published Online September 17, 2010
F. Other diseases characterized by increased osteoclast activity
VI. Drug-Induced Osteoclast Subtypes
A. Existing drugs
B. Future treatments
VII. The Bone Anabolic Effects of the Osteoclasts
VIII. Conclusions and Future Perspectives
I. Introduction
steoclasts are multinucleated bone-resorbing cells
that are unique in their ability to degrade mineralized matrices, such as bone and calcified cartilage (1). Osteoclasts have for a long time been considered boneresorbing “machines,” yet some years ago it was
demonstrated that not all osteoclasts are the same and that
careful elucidation of the osteoclast subtype may prove
O
Abbreviations: AGE, Advanced glycation end-product; BMD, bone mineral density; CAII, carbonic anhydrase II; ClC-7, chloride channel 7; CT-1, cardiotrophin-1; CTX, C-terminal
crosslinked telopeptide of type I collagen; ER, estrogen receptor; GLP, glucagon-like peptide;
HRT, hormone replacement therapy; ICTP, carboxyterminal peptide of type I collagen; MMP,
matrix metalloproteinase; ONJ, osteonecrosis of the jaw; OPG, osteoprotegerin; RA, rheumatoid arthritis; RANK, receptor activator of nuclear factor ␬B; RANKL, RANK ligand; SERM,
selective estrogen receptor modulator; TRACP, tartrate-resistant acid phosphatase; V-ATPase,
vacuolar type ATPase.
Endocrine Reviews, February 2011, 32(1):31– 63
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Henriksen et al.
Osteoclast Subtypes
beneficial (1– 4). As illustrated in Table 1, under normal
circumstances, osteoclasts are influenced by a complex
combination of systemic hormones and local mediators
present in the different bones. Under pathological conditions, such as cessation of estrogen production or inflammatory conditions, additional cytokines are present. Even
the bone type and age may influence the phenotype of the
osteoclast (2, 5–7). Finally and importantly, different
classes of drugs used for treatment of osteoporosis and
other diseases also influence the osteoclasts significantly.
The aim of this review is to provide a thorough description of the complex nature of osteoclasts under healthy
and diseased states and to describe their modulation by
drugs that have been approved for use or are under development. The paper will also emphasize the role of osteoclasts in initiating bone formation, a recently discovered activity of these cells that has gained much attention
(3, 8 –13).
II. Bone Remodeling
Bone remodeling is required for optimal control of calcium homeostasis and strength of the bones and is essential
for the continued maintenance of a healthy skeleton (14).
Bone remodeling is performed by three cell types: 1) the
osteoclasts, which are the sole cells in the body possessing
the ability to degrade both the inorganic calcium matrix
and the organic collagen matrix; 2) the osteoblasts, which
are the bone-forming cells; and 3) the osteocytes, which
appear to regulate the activity of both osteoclasts and osteoblasts (14, 15). In healthy adults, under normal circumstances, bone resorption is always followed by an
equal degree of bone formation, a tightly balanced process
referred to as coupling (9, 16). The modulation of activities of the cells involved in the remodeling cycle was recently described in detail (15).
Coupling was initially discovered in the 1960s when
Frost and co-workers (17, 18) demonstrated that osteoblasts filled the resorption pits created by osteoclasts in
more than 97% of the cases (17–21). Since then, coupling
has been understood as a coordinated and balanced induction of osteoblastic bone formation in response to
prior bone resorption (19). Uncoupling occurs when the
balance between resorption and formation is disrupted,
which often leads to pathological situations such as osteoporosis or osteopetrosis (3, 9, 14, 22, 23). However,
uncoupling also occurs under physiological conditions,
i.e., during skeletal growth in children, where bone formation exceeds bone resorption (10).
Hypogonadal osteoporosis is usually caused by a decrease or loss of sex steroid production, which results in
accelerated osteoclastogenesis and bone resorption (24)
Endocrine Reviews, February 2011, 32(1):31– 63
that cannot be completely countered by an increase in
bone formation. This results in low bone mass, in deterioration of the microarchitecture of the skeleton, and often
in fractures (25). Osteoporotic fractures are associated
with increased morbidity and mortality and give rise to a
significant public health problem (24).
Osteopetrosis, on the other hand, is a rare, inherited
disease in various species including man, which was originally identified by Albers-Schönberg in 1904 (26). In the
majority of cases, it is caused by defective resorption by the
osteoclasts, resulting in high bone mass with poor bone
quality and increased fracture frequency due to defective
bone remodeling (1, 26, 27). However, recent studies also
characterized patients with osteopetrosis due to dysfunctional osteoclastogenesis either directly affecting
the osteoclast precursors or indirectly through the osteoblasts, and thus the phenotype was caused by the
absence of osteoclasts, rather than inactivity of these
cells (28 –31). Interestingly, the studies of osteopetrotic
patients have indicated that the presence of osteoclasts,
but not their activity, is essential for bone formation,
indicating that some aspects of the coupling principle
should be revised (1, 3).
Because hypogonadal osteoporosis is associated with
increased numbers and activity of osteoclasts (16), most
treatments developed so far, such as bisphosphonates and
hormone replacement therapy (HRT)/selective estrogen
receptor modulators (SERMs), have focused on eliminating or reducing the number of osteoclasts and thereby
reducing bone resorption (32). These treatments are associated with secondary decreases in bone formation due
to the coupled nature of the bone remodeling process,
which naturally limits their efficacy (3, 24). However, as
seen in the osteopetrotic syndromes, there are indications
that bone resorption and bone formation can be dissociated, and from recent studies it appears that the osteoclast
itself, whether it is a physiological, pathological, or druginduced subtype, is highly important for a secondary effect
on bone formation (1, 3).
In this review, we describe differences in osteoclast
activity and subtypes in relation to physiology, pathophysiology, and medication, with special attention to
coupling in the bone remodeling process. Ultimately,
this review highlights potential directions for new treatment modalities.
III. The Classical Osteoclast
Osteoclastogenesis is a complex process requiring both the
correct extracellular stimuli and the correct cellular molecules to interact without impediment (22, 33). Osteoclasts
arise from hematopoietic stem cells that, in the presence of
Endocrine Reviews, February 2011, 32(1):31– 63
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33
TABLE 1. A simplified summary of osteoclast phenotypes as a function of physiology, pathology, and drugs, also
indicating areas of osteoclast biology that are not well-understood
Osteoclast no.
Bone resorption
Classical osteoclast
Physiology
Targeted
Bone formation
Resorptive process
Acid
Cat K
MMP
⫹⫹⫹
⫹⫹⫹
⫹/⫺
Balanced
Balanced
Normal
Normal
Recruitment to specific
areas increased
Not clear
Normal
Normal
Normal
Normal
Normal
Increased
⫹⫹
?
?
Not clear
Increased
Decreased
Normal
Normal
Normal
⫹
⫹⫹
⫹
⫹
⫹
⫹
?
⫹⫹
?
⫹
⫹
⫺
?
⫹/⫺
?
⫹⫹
⫺
⫹
Normal
Normal
Normal
Normal
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫺
⫺
Increased
Increased
⫹ ⫹ ⫹⫹
⫺
Age
OC-rich OP
Increased
Greatly increased
Increased
Decreased
⫹⫹
⫺
⫹⫹
⫺
?
⫺
OC-poor OP
Pycnodysostosis
No osteoclasts
Unchanged/increased
osteoclast size
Greatly increased at
local sites
Decreased
Decreased
⫺
⫹
⫺
⫺
⫺
⫹⫹⫹
⫹⫹
⫹
⫹⫹⫹
Greatly increased at
local sites
Greatly increased at
local sites
Not known
⫹
⫹
⫹⫹⫹
Increased at local sites
⫹
⫹
⫹⫹⫹
Drug-induced
BPs
Reduced
Decreased
⫺
⫺
?
HRT/SERMs
Reduced
Decreased
⫺
⫺
?
Calcitonin
Unchanged
Decreased
⫺
⫺
?
PTH
Increased/unchanged
Decreased
⫹⫹
⫹⫹
?
Strontium ranelate
GCs
Denosumab
Unchanged
Unchanged/increased
Greatly reduced
Decreased?
Unchanged/increased
Decreased
⫺
⫹
⫺
⫺
⫹
⫺
?
?
⫺
Cat K inhibitors
Unchanged
Decreased
⫹
⫺
⫹⫹⫹
GLP-2
Unchanged
Decreased
⫺
⫺
?
Acidification inhibitors
Increased
Decreased
⫺
⫺
⫺
Stochastic
Night
Day
Chondroclast
Endochondral
Intramembraneous
Trabecular
Cortical
Pathology
Osteoporosis
Paget’s
RA
Lytic metastases
Increased at local sites
Not clear
Minor up-regulation
Minor down-regulation
Balanced
Balanced
Not balanced, opposite
side of bone than
resorption
Balanced
Balanced
Increased, but less than
resorption
Decreased
Increased according to
increased OC
number
Decreased?
Not clear
Increased locally, but
does not
compensate
resorption
Not known
Increased locally, but
does not
compensate
resorption
Decreased secondary
to resorption
Decreased secondary
to resorption
Not changed or minor
decrease
Increased, but only
temporarily
Increased
Decreased strongly
Decreased secondary
to resorption
Decreased secondary
to resorption
Not changed, but long
term effects are not
known
Increased, but so far
only in animal
models
The table shows the subtype of osteoclasts, the number of osteoclasts, the effect on bone resorption, which part of the resorption machinery that is active/affected,
and the effect on bone formation. OC, Osteoclast; OP, osteopetrosis; Cat K, cathepsin K; BPs, bisphosphonates; GCs, glucocorticoids; MMP, matrix metalloproteinase;
RA, rheumatoid arthritis; PTH, parathyroid hormone; HRT, hormone replacement therapy; SERMs, selective estrogen receptor modulators; GLP-2, glucagon-like peptide 2.
34
Henriksen et al.
Osteoclast Subtypes
Endocrine Reviews, February 2011, 32(1):31– 63
FIG. 1. Schematic illustration of the molecules involved in osteoclastogenesis and function. (See Refs. 1, 33, and 42). NF␬B, nuclear factor ␬ B;
TRAF6, TNF receptor-associated factor 6; nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1; PYK2, Proline-rich Tyrosine
Kinase 2; MitF, microphthalmia-associated transcription factor; ClC-7; Chloride Channel 7; PLEKHM1, pleckstrin homology domain containing,
family M (with RUN domain) member 1; osteopetrosis associated transmembrane protein 1; CA2, carbonic anhydrase II; AE2, anion exchanger 2.
receptor activator of nuclear factor ␬B (RANK) ligand
(RANKL) and macrophage-colony stimulating factor, undergo differentiation and fusion resulting in large multinucleated cells characterized by expression of a series of osteoclast markers, such as tartrate-resistant acid phosphatase
(TRACP), matrix metalloproteinase (MMP)-9, cathepsin K,
carbonic anhydrase II (CAII), the a3 subunit of the vacuolar
[H⫹]-ATPase, chloride channel 7 (ClC-7), osteopetrosis-associated transmembrane protein 1, and the calcitonin receptor (34 – 41). Osteoclastogenesis and the molecules involved
in this process are summarized in Fig. 1, but are not
discussed in any further detail because several excellent
reviews have been published recently on this topic (1,
22, 33, 42).
Polarization and formation of the sealing zone, which
is a specialized ring structure containing a high number of
␤-actin filaments, are the next steps in the life span of an
osteoclast (43, 44). These processes require the ␣v␤3 integrin and the intracellular signal transducers c-src, Syk,
and proline-rich tyrosine kinase 2, as well as the microphthalmia transcription factor (33, 45, 46), which appears to
be an important regulator of osteoclastic gene transcription (47– 49).
The final step of osteoclastogenesis is the activation of
resorption, a process that is characterized by the formation of a ruffled border that is an intensely convoluted
membrane present inside the sealing zone (43, 44). The
formation of the ruffled border is not well-characterized;
however, the signaling molecule Rab7 is required (50).
Bone resorption takes place at the ruffled border localized at the apical side of the osteoclasts, and it can be
divided into two processes, namely acid secretion and pro-
teolysis, although these processes likely occur at the same
time (44, 51).
Bone resorption is initiated by active secretion of protons through a vacuolar type ATPase (V-ATPase) and passive transport of chloride through a chloride channel (52,
53). The secretion of hydrochloric acid leads to dissolution
of the inorganic matrix of the bones (54). The osteoclastic
V-ATPase is functionally specific and contains the a3 subunit, and accordingly, loss of a3 leads to osteopetrosis (37,
55–57). In both mice and man, the chloride channel ClC-7,
has been shown to mediate chloride transport, thereby
ensuring the electrochemical balance required for intense
acidification (Fig. 2) (8, 39, 58). Recent data showed that
ClC-7 functions as a proton-chloride antiporter (59, 60).
To generate the necessary levels of H⫹ and Cl⫺, the
enzyme CAII catalyzes conversion of CO2 and H2O into
H2CO3, which ionizes into H⫹ and HCO3⫺ (35), thereby
providing the protons for the V-ATPase (27). Meanwhile,
basolateral exchange of HCO3⫺ ions for Cl⫺ by anion exchanger 2 (61– 63) provides Cl⫺ ions required for the intense acidification occurring in the resorption lacuna.
Interestingly, long bones differ from flat bones with
respect to the molecular nature of the acidification machinery (62).
Proteolysis of the type I collagen matrix in bones is
mainly mediated by the cysteine proteinase, cathepsin K.
This enzyme is active at low pH in the resorption lacuna
(Fig. 2) (64 – 68). The neutral MMPs also appear to play a
minor role during organic matrix degradation; however,
the exact role of MMPs is still being investigated (69) and
is highly dependent on the bone type (38, 70, 71). The
resorbed material is removed from the resorption pit by
Endocrine Reviews, February 2011, 32(1):31– 63
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35
FIG. 2. Top, Schematic illustration of the differences between acid secretion and proteolysis during osteoclastic bone resorption, illustrating that
the collagen matrix is removed by proteolysis after acidification. Bottom, Mutations/knockout in genes/proteins involved in bone resorption,
phenotypes, and effect on osteoclasts. Ae2, Anion exchanger 2; a3 V-ATPase, a3 subunit of the osteoclast-specific V-ATPase. ClC-7, chloride
channel 7; OSTM1, osteopetrosis associated transmembrane protein 1; CA2, carbonic anhydrase 2; MMP-9, matrix metalloproteinase 9.
uptake and transcytosis through the osteoclast (72, 73).
After completing resorption, osteoclasts either undergo
apoptosis or perform a further round of resorption (44)
(Table 1).
In summary, the osteoclasts are highly specialized for
both dissolution of the inorganic matrix and degradation
of the organic matrix of the bones. These highly polarized
cells are characterized by a unique set of membrane-bound
molecules that ensure an efficient resorption of bone and
other mineralized tissues. This complex machinery may be
affected by a range of important parameters in physiology
and pathology, and importantly in drug-induced situations that are important to identify and advance osteoclast
research and biology.
IV. Osteoclast Subtypes in
Physiological Situations
Osteoclast activities are essential for development, as well
as remodeling of bone in response to aging and stress (6,
14, 15). Under normal physiological circumstances, the
osteoclasts can be categorized into subgroups depending
on the matrix on which they are positioned, the time of
day, and the type of remodeling in which they participate.
These different groups of osteoclasts have provided key
information on skeletal maintenance.
A. Endochondral vs. intramembranous bone osteoclasts
Anatomically, two types of bones are present in the
body, the long bones (e.g., the femur and tibia) and the
36
Henriksen et al.
Osteoclast Subtypes
flat bones (e.g., the calvarium), with the main difference
between these two types of bone being their development (74). Studies also indicate that osteoclasts on these
two types of bones are functionally different with respect to both acid secretion and proteases involved in
degradation (2).
Evidence that differences between resorption of flat and
long bone exist was presented in 1999 (4). However, indications that even the acidification process is different
have been published only recently (62). Data from mice
deficient in the bicarbonate-anion exchanger Ae2 (Slc4a2)
have shown that it is essential for bone resorption in long
bones (61, 63), whereas it is not involved in bone resorption in calvariae (62), showing that distinct acid transport
mechanisms are present in different subsets of osteoclasts.
With respect to acid secretion into the resorption lacunae,
it is presently not known whether any differences exist,
although the absence of calvarial thickening in patients
with defective ClC-7 strongly suggests that ClC-7 is not
involved in resorption of the flat bones (75).
Extensive research into the proteolytic processes involved in resorption of flat and long bones clearly demonstrates that the proteolytic processes involved in degradation of these two types of bone matrix are distinct (4,
71). Osteoclasts in flat bones preferentially appear to engage in MMP-mediated bone resorption, although cathepsin L seems to be involved, too. Osteoclasts in long bones
primarily depend on cysteine proteinases, in particular cathepsin K (4, 71). TRACP also appears to be involved in
bone resorption, and more so in calvarial bone (76 –78).
When osteoclasts generated from human peripheral blood
are seeded on cortical bone, they primarily depend on cathepsin K, whereas when cathepsin K activity is blocked,
there is some compensatory bone resorption mediated by
MMPs (69). How these osteoclasts behave on bone substrates other than cortical bone is presently not known
(Table 1).
Data suggesting that the bone matrix could play a role
in the control of osteoclastic activities were presented in a
study showing compositional differences between long
bone and flat bone matrices, including differences in the
presence of putative cysteine proteinase inhibitors (79).
The functional significance of these data still remains to be
fully elucidated, although they clearly illustrate the importance of understanding how a given context affects the
osteoclasts.
B. Chondroclasts
It has long been discussed whether chondroclasts are a
“real” cell type or whether they simply are osteoclasts that
reside on cartilage instead of bone (77, 80 – 82). Chondroclasts are mainly important in endochondral bone development, and in addition there is some evidence that
Endocrine Reviews, February 2011, 32(1):31– 63
chondroclasts may also play a role in both rheumatoid
arthritis (RA) and osteoarthritis (83– 85). The term chondroclast derives from the localization of these cells on calcified cartilage as seen in the expanding growth plates
during endochondral ossification (80, 86). For their formation, these cells are dependent on the presence of
macrophage-colony stimulating factor and RANKL, as
are bone-resorbing osteoclasts (87–90).
Most of the evidence for the functionality of chondroclasts is derived from studies of the longitudinal growth of
long bones, i.e., metatarsals and tibias isolated from
mouse embryos (38, 80, 91–93). First, bone/cartilage resorption in these models is still dependent on acid secretion
as evidenced by mice with mutations in the acid secretion
process, i.e., ClC-7-deficient and a3 V-ATPase-deficient
mice, as well as mice unable to form sealing zones, i.e.,
c-src-deficient mice, in which the massive bones mainly
consist of calcified cartilage due to the defective resorption
process (56, 94, 95). Similar findings have been noted in
the corresponding human disease(s) (27, 96, 97). Interestingly, the ruffled border is less prominent in the chondroclasts than osteoclasts, potentially suggesting that lower
levels of acid secretion are required for dissolution of this
matrix (98, 99). The main difference between chondroclasts and osteoclasts is in the profiles of enzymes necessary for tissue degradation. Resorption by chondroclasts
does not appear to depend as much on cathepsin K as does
resorption by osteoclasts. Cathepsin K-deficient mice
show no evidence of calcified cartilage in the marrow cavity of long bones, indicating that the removal of calcified
cartilage during endochondral ossification occurs, although there are indications that the process is delayed
(65, 100). Of importance is the observation of a massive
compensation by MMPs in the absence of cathepsin K,
which obscures the interpretation of data from cathepsin
K-deficient systems (69, 70, 101–103). Finally, one study
has indicated that TRACP is activated and secreted at the
ruffled border in cathepsin K-deficient osteoclasts, and
only in cells that play a role in the removal of calcified
cartilage (104) (Table 1).
Although chondroclasts are involved in the degradation of a different matrix than osteoclasts, an interesting
observation is that bone formation is tightly coupled to
resorption of the mineralized matrix, as has clearly been
demonstrated in studies of endochondral ossification (86).
It is more likely that bone formation is coupled to chondroclast numbers because release of molecules from degradation of cartilage, which in composition is far from
bone, would be expected to be different from molecules
released during bone resorption; however, this has never
been studied in detail.
Endocrine Reviews, February 2011, 32(1):31– 63
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FIG. 3. Schematic illustration of stochastic vs. targeted remodeling. The figure illustrates the local nature of targeted remodeling, which is
activated at specific sites after the formation of microcracks and leads to removal of the microcrack and restoration of the damaged bone.
Stochastic remodeling, on the other hand, is of a systemic nature and is activated by low calcium levels in the circulation leading to PTH release.
Other hormones, such as vitamin D3 (VitD3), and potentially calcitonin also play roles in stochastic remodeling. It appears that there are two levels
of calcium homeostasis— one mediated by osteocytes independent of osteoclasts, and one including the osteoclasts—although the balance
between these two ways of releasing calcium still remains to be fully understood.
C. Osteoclasts involved in targeted and
stochastic remodeling
Two different modes of remodeling have been proposed: targeted and stochastic. Targeted bone remodeling
takes place at specific sites, whereas stochastic remodeling
occurs more randomly (19, 105, 106). The first type is
primarily performed to replace microdamaged bone and
thus to maintain the load-bearing capacity of the skeleton.
The second type of remodeling appears random with respect to localization, although it may be involved in maintaining integrity of the bones, independent of damage.
This process is hormonally regulated (105, 107).
The balance between these two modes of bone remodeling has not been fully elucidated yet, but studies in dogs
indicate that approximately 30% of all remodeling is targeted and the remaining 70% is stochastic (105). With
respect to the osteoclasts mediating these two types of
remodeling, most studies have focused on how targeted
remodeling is controlled.
It appears that the bone matrix contains signals regulating the activity of the osteoclasts (108). A recent study
demonstrated that aged bones were more readily resorbed
than young bones, thus supporting the hypothesis that the
bone matrix composition influences remodeling rates (7).
Furthermore, areas of microdamage, which are character-
ized by high numbers of apoptotic osteocytes, are preferentially and rapidly degraded by osteoclasts. This further
supports the possibility that changes in the bone matrix
and the balance between live and dead osteocytes determine which areas will be remodeled (108). Finally, a recent
study demonstrated that targeted ablation of osteocytes
led to a dramatic increase in osteoclast activity (109) (Table 1). These data indicate that death of osteocytes is a key
point in induction of osteoclast activity (Fig. 3).
Taken together with the finding that bones of different
age lead to different levels of osteoclastogenesis (7), it appears that osteoclast functionality is at least partially controlled by osteocyte-derived molecules, which are sequestered in the bone matrix.
Stochastic remodeling, while occurring at random sites,
is centrally regulated by hormones such as PTH, vitamin
D3, and potentially calcitonin, and its main role is the
regulation of calcium homeostasis (110 –112). It has even
been questioned to what extent this process depends on the
presence of osteoclasts because patients with nonfunctional osteoclasts have normal calcium homeostasis (110 –
112). Yet, when they are calcium-deprived, osteopetrotic
patients fail to correct their calcium levels, indicating that
osteoclasts, which are either absent or nonfunctional in
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Osteoclast Subtypes
these patients, do play a role in calcium homeostasis, and
thus stochastic remodeling (Fig. 3) (113).
In summary, targeted remodeling is beginning to be
understood in detail, and it is a tightly regulated and coupled process involving osteocytes, osteoclasts, and cells of
the osteoblast lineage. On the other hand, stochastic remodeling and the role it plays in calcium homeostasis are
still not very well understood, although there are indications that there is a level of regulation by the activity of
osteoclasts.
D. Trabecular and cortical osteoclasts
Bone remodeling does not occur with the same frequency in cortical and trabecular bone. Every year, 25%
of the trabecular bone matrix, but only 4% of the cortical
matrix, is remodeled (114). Interestingly, most in vitro
osteoclast experiments are based on cortical bone (or dentine) substrates, which are either slowly remodeled or not
remodeled at all (7, 102, 114 –116). Studies have shown
that bones endogenously contain signals regulating osteoclastogenesis and resorption and that these signals appear
to be related to the age of the bone (7, 117) (Table 1). Thus,
an interesting question is whether osteoclasts themselves
are indeed different when derived from different matrices
or whether the difference is matrix related. Furthermore,
systemic regulation is likely to be involved in controlling
which bones are resorbed to some extent.
These data also correlate with evidence indicating that
remodeling of different bone compartments can be either
primarily targeted, such as in the cortex, or primarily stochastic, as seen in some parts of trabecular bone (106). A
further understanding of this could provide directions for
the development of novel drugs producing optimal benefit
at the sites where it is most needed, i.e., leading to a better
fracture reduction than that presently obtained.
E. Diurnal variation in osteoclasts or osteoclast activity?
Bone resorption markers measured in serum may be
interpreted as indicating the net result of all osteoclast subtypes and activity levels at one particular time. A wide range
of factors, known and unknown, may influence the interpretation (15). Diurnal variation is a well-established and
important parameter of bone turnover. Postprandially, bone
resorption decreases by approximately 50% compared with
that of fasting individuals, but during the night, bone resorption increases to an equally large degree (118 –120). Several
investigations have demonstrated that the circadian variation in bone resorption is induced in part by food intake
(121–123), which, at least partially, involves the peptide hormone glucagon-like peptide (GLP) 2 (124). Interestingly, the
osteoclast number does not appear to depend on the time of
day, further emphasizing differences between osteoclast
Endocrine Reviews, February 2011, 32(1):31– 63
number and activity (32) (Table 1). An interesting aspect of
this is that targeting nocturnal resorptive activity appears to
lead to inhibition of bone resorption, whereas not attenuating bone formation (125–127), thereby highlighting an interesting prospect of reducing bone resorption in a specific,
nocturnal manner.
In summary, studies of osteoclasts under different physiological conditions, such as those listed above, have highlighted the heterogeneity of these cells. Furthermore, these
studies highlighted the importance of the balance between
bone resorption and bone formation, a tightly regulated
phenomenon that rarely is disturbed under physiological
conditions. Finally, how the heterogeneity of the osteoclasts affects bone formation is presently not well understood, but a further understanding of this process could
help optimal treatment of diseases involving alterations in
bone remodeling.
V. Osteoclast Subtypes in
Pathological Situations
Changes in osteoclast activity and number have been detected in several diseases, ranging from illnesses involving excessive bone resorption, such as osteoporosis and
Paget’s disease; to those involving secondary activation of
osteoclasts, such as osteolytic metastases and RA; to diseases involving defective osteoclast differentiation and/or
function, such as osteopetrosis. These different types of
diseases have shed important light on osteoclastic function
with respect to obtaining the right type of treatment. They
have also shed light on a very central aspect in bone biology, the coupling principle. The coupling principle describes the phenomenon that bone formation follows bone
resorption, which leads to a complete restoration of the
bone removed during bone resorption (17).
A. Osteoporotic osteoclasts
1. Changes in osteoclastogenic potential in osteoporosis
An important aspect of osteoporosis is whether the
number of osteoclast precursors in the circulation increases, and, if so, whether the osteoclastogenic potential
of these cells is increased. Eghbali-Fatourechi et al. (128)
showed that the overall number of cells expressing
RANKL is increased in postmenopausal women compared with premenopausal or estrogen-treated women,
clearly indicating that the bone marrow microenvironment, including stromal, T, and B cells, changes in a proosteoclastic direction when estrogen is reduced. These data
were supported by a recent study from the same authors
showing that bone marrow cells isolated from estrogentreated or control postmenopausal women displayed
Endocrine Reviews, February 2011, 32(1):31– 63
reduced osteoclastogenic potential (129) (Table 1). Estrogen was shown to have a dual mode of action—the
first leading to overall lower RANKL expression by
bone marrow cells, and the second reducing the osteoclastogenic response to RANKL (130). Interestingly,
aging of mice was also shown to increase the osteoclastogenic potential of bone marrow cells, both by upregulation of RANKL production and by increasing
precursor sensitivity to RANKL (131).
In vitro studies of the changes in cellular activity of
osteoclasts from osteoporosis patients are limited, but
these have indicated both an accelerated osteoclastogenesis and resorption (132, 133). Furthermore, a key cell in
the up-regulation of osteoclastogenesis appears to be the
T cell, which responds to lowered estrogen levels by increasing RANKL production (134).
The main issue with all the studies of osteoporotic osteoclasts and their precursors is the use of mixed cell populations, which clouds the interpretation of the results,
and therefore these aspects of osteoclastic function still
require further investigation. Furthermore, with the recent
publication of the possibility of assessing the “anabolic
potential” of osteoclasts (11, 13), it would be of interest to
investigate the anabolic capacity of osteoporotic osteoclasts and thus shed light on the imbalance between bone
resorption and bone formation in osteoporosis.
2. A direct role for sex steroids on osteoclasts
The role of estrogen on cells belonging to the osteoclastic lineage has been studied extensively, with several findings indicating that estrogen suppresses osteoclastogenesis
but not the resorptive activity of mature osteoclasts (135–
137). Androgens, such as dihydrotestosterone, exhibit
similar effects to estrogen on osteoclasts in vitro, although
this has not been studied in great detail (138). Finally, a
recent study using mice deficient for the estrogen receptor
(ER)-␣, specifically in mature osteoclasts, showed bone
loss in female, but not male, mice (139). This demonstrated that estrogen likely plays a direct role in bone resorption by even mature osteoclasts (139). Although the
authors used the cathepsin K promoter to ensure specific
knock-down of the ER-␣ in osteoclasts, cathepsin K is also
expressed in preosteoclasts, albeit to a lower extent (139).
More studies are needed to investigate the role of ER-␣ in
mature osteoclasts specifically. Interestingly, osteoclasts
in different bone sites preferentially express different ERs,
with cortical osteoclasts mainly expressing ER-␣ and
trabecular osteoclasts mainly expressing ER-␤ (140),
whereas from a functional point of view ER-␣ appears to
be more relevant for trabecular, not cortical, bone (141).
Furthermore, the expression pattern also differs between
mature and differentiating osteoclasts; ER-␣ is mainly expressed in immature cells, and ER-␤ is present at all stages
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of osteoclastogenesis (137). Again, there appear to be different osteoclastic subtypes, which also appear to be relevant in the context of bone loss rates in different bone
compartments during osteoporosis (114). As indicated
above, an important point is the difference between genders (139). In mice, the gender-based difference between
cortical apposition and endocortical resorption that becomes more apparent with increasing age might be explained by differences in ER expression (142). With respect to changes in the osteoclasts after menopause, a
couple of studies have clearly demonstrated that bone resorption, as well as bone formation, increases in women
after menopause (143–145), and these changes become
more explicit in high- and low-turnover patients (145).
Although bone formation increases as a function of the
increased resorption, it does not match bone resorption,
thereby illustrating the importance of understanding the
interplay between osteoclasts and osteoblasts in detail.
B. Changes in osteoclast activities with increasing bone
matrix age
Numerous studies have investigated the control of osteoclast activity as a function of changes in biochemical
properties of the bone matrix. Aging leads to accumulation of different biochemical modifications of the bone
matrix, such as advanced glycation end-products (AGEs),
homocysteine, increased calcium concentration, as well as
some modifications of the collagen matrix (146).
Recent studies have indicated that these modifications
of the bone matrix itself actually modulate the activity of
the osteoclasts to a certain extent (7, 117). Homocysteine,
which accumulates in bone and in circulation with age,
was shown to activate osteoclastogenesis and bone resorption (147) (Table 1). AGEs are modifications of proteins
that accumulate in various tissues with age, and they have
been implicated in the pathology of osteoporosis (146,
148). Some evidence indicating a direct regulation of osteoclast activity by AGEs has been published, but these
studies are contradictory. One study shows activation of
resorption by AGE-modified proteins (149), whereas the
other study shows the opposite (117); however, quite different techniques were used.
Interestingly, AGEs are accumulated in diabetes, and
they have been speculated to be involved in the increased
fracture rates observed in patients with this disease (150,
151). Another intriguing finding is the induction of
apoptosis in osteoblasts by AGEs (152), which potentially
could play a role in the imbalance between osteoclast and
osteoblast function during osteoporosis and aging. These
findings are all preliminary in nature, and they await confirmation from independent research groups. However,
once again they illustrate the heterogeneity of osteoclasts,
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Henriksen et al.
Osteoclast Subtypes
in this case as a function of matrix age, and the importance
of understanding this phenomenon.
C. Osteoclast-rich osteopetrosis
The most frequently occurring forms of osteopetrosis
are those caused by mutations in either the a3 subunit of
the V-ATPase, ClC-7, or osteopetrosis-associated transmembrane protein 1. Osteoclasts from patients with mutations in these genes or proteins and from knockout mice
have been studied quite extensively (8, 39, 56, 58, 95,
153–156).
Microscopic analyses of cells from patients with defective acid secretion by osteoclasts indicated defective ruffled border formation, but also accumulation of material
in vesicles, indicating hampered transcytosis (157). Apart
from confirming the defective acid secretion and thereby
bone resorption, when either ClC-7 or the a3 subunit is
mutated (8, 39, 56, 58, 95, 153, 154), these studies also
shed light on important aspects of bone remodeling.
In vitro studies indicate that osteoclasts with impaired
acid secretion have higher survival rates than cells with
normal secretion, due to the reduced release of proapoptotic signals during resorption (159). This observation
correlates well with the high numbers of osteoclasts observed in vivo in this group of patients, as well as with
findings in mice with attenuated acidification of the resorption lacunae (95, 97) (Table 1). Furthermore, significantly increased resorbed areas are seen during impaired
acid secretion, but the resorption pits are shallow, indicating a disturbed activity of the osteoclasts (97). More
importantly, these studies highlighted that bone formation in these patients is ongoing—a process that appears to
be correlated to the increased number of osteoclasts rather
than bone resorption (96, 154, 160, 161). These findings
contrast with the classical perception that bone formation
always follows bone resorption in a tightly coordinated
manner and illustrate the importance of the actual presence of osteoclasts to maintain bone formation.
D. Osteoclast-poor osteopetrosis
Several murine forms of osteoclast-poor osteopetrosis
have been described in the literature (1, 42). In general,
whereas the mutations express a pronounced osteopetrotic phenotype and few or no osteoclasts are present, the
phenotypes are less severe than the phenotypes of the different osteoclast-rich osteopetrotic mutations (1). These
data strongly suggest that the osteoclasts are indeed involved in the production of anabolic signals for bone formation (3, 15).
Studies of mice deficient in c-src and c-fos, a key molecule involved in ruffled border formation and a key signal
transducer for osteoclastogenesis, clearly demonstrated
Endocrine Reviews, February 2011, 32(1):31– 63
that osteopetrosis was due to nonfunctional osteoclasts or
the absence of osteoclasts, respectively (162, 163). Interestingly, these two groups of mice have opposing phenotypes with respect to bone formation. The osteoclast-rich
c-src knockouts have increased bone formation (164), and
the osteoclast-poor c-fos knockouts have decreased bone
formation (165). The anabolic effects of PTH are present
in the c-src⫺/⫺ mice but are blunted in the c-fos⫺/⫺ mice
(166), indicating that osteoclasts are central for bone formation (Table 1).
Two recent studies identified mutations in the genes for
RANK and RANKL as the causes of osteopetrosis in a
novel group of patients (28, 29). No indications of osteoclasts were found in these patients (28, 29), which is consistent with previous observations in mice deficient in both
RANKL and RANK (87, 90). Patients with mutations in
either RANKL or RANK have a pronounced osteopetrotic
phenotype and classical histological hallmarks of osteopetrosis including unresorbed primary spongiosa. However, although limited data have been published, the osteopetrotic phenotype appears to be less severe than the
one observed in the osteoclast-rich forms (1). Thus, mutations within the RANK/RANKL/osteoprotegerin (OPG) system can lead to osteoclast-poor osteopetrosis with low bone
formation in mice and men.
Interestingly, alterations in osteoblast function, such as
changes in the production of RANKL and OPG, may have
the same effect. Stabilizing osteoblastic ␤-catenin in transgenic mice, thus mimicking constitutive activation of the
canonical Wnt signaling pathway, was followed by an
up-regulation of OPG in relation to RANKL (31, 167). As
expected, the mice developed osteoclast-poor osteopetrosis with failure of tooth eruption, a classical phenomenon
in murine osteopetrosis. Mutations within LRP5 related
to the Wnt signaling pathway have underscored the fundamental importance of this pathway for regulation of
bone mass. The osteoporosis pseudoglioma syndrome was
found to be caused by loss of function mutations in the
gene for LRP5 (168). In contrast, mutations affecting the
first propeller of the coreceptor, presumed to be followed
by chronic activation of the Wnt pathway, were found in
various forms of monogenic human osteosclerotic phenotypes (169). Among these, autosomal dominant osteopetrosis type 1 has been well characterized clinically, biochemically, histomorphometrically, and biomechanically
(75). Autosomal dominant osteopetrosis type 1 is an osteoclast-poor osteopetrotic phenotype with increased biomechanical competence and no low-energy fractures.
Osteoclast profiles are markedly decreased (97), bone
formation seems to be normal, and OPG levels in the circulation increased (170). However, when investigating os-
Endocrine Reviews, February 2011, 32(1):31– 63
teoclasts ex vivo from these patients, they express normal
bone resorptive capacity (30).
In summary, osteoclast-poor osteopetrosis can arise in
murine mutations/transgenics or humans when the OPG/
RANKL/RANK system is affected directly or indirectly.
These findings underscore this cytokine system as a key
regulator of osteoclastogenesis. Moreover, the phenotypes seem to be less affected than the osteoclast-rich
forms, the reason for which is so far unresolved, although
there are indications that reductions in bone formation are
involved (1, 28, 29).
E. Pycnodysostotic osteoclasts
An interesting subtype of osteoclasts with defective
bone resorption is observed in patients with pycnodysostosis. Pycnodysostosis is caused by loss of function or loss
of expression mutations in the cysteine proteinase cathepsin K, which in humans causes dwarfism and poor bone
quality due to defective remodeling of the bones (36, 171–
173). Few studies examining the phenotype of pycnodysostotic osteoclasts have been published. Microscopic analyses of the osteoclasts have shown significantly increased
amounts of demineralized collagen matrix in the resorption pit, but also inside the osteoclasts, indicating disturbed resorption and trafficking of resorbed components
(174, 175). A study of biochemical markers of bone turnover showed that C-terminal crosslinked telopeptide of
type I collagen (CTX-I) release was reduced, whereas production of the MMP-generated type I collagen fragment
carboxyterminal telopeptide of type I collagen (ICTP) was
increased (67) (Table 1). Several studies of cathepsin Kdeficient mice have been published, and whereas they confirm that cathepsin K is essential for degradation of the
organic matrix in bone (65, 66, 176), there are also several
differences between the human and mouse phenotypes
(103). Furthermore, in cathepsin K-deficient mice, bone
formation parameters are highly increased (100). These
findings have not been replicated in pycnodysostosis patients in whom the bone matrix is disordered (177), and a
clinical case study indicated that anabolic response to PTH
was absent (174). Two recently published clinical studies
have shown that whereas bone resorption markers are
strongly reduced, bone formation is also suppressed in
women treated with the cathepsin K inhibitor odanacatib
(178). In a monkey study monitoring bone formation by
histomorphometry reductions in bone formation, rates
were shown in the trabecular bone compartment, whereas
bone formation was increased in the cortical compartment
(179 –181).
In conclusion, cathepsin K mediates cleavage of type I
collagen in the resorption lacunae, but its secondary effects on bone formation are bone type-dependent and still
need to be investigated further.
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F. Other diseases characterized by increased
osteoclast activity
Apart from hypogonadal osteoporosis, several diseases
are characterized by accelerated osteoclastogenesis and
function. Although the etiology of these diseases is different, there are interesting overlaps and discrepancies that
provide highly useful information about osteoclastic function and secondary effects on bone formation under different circumstances (182).
1. Pagetic osteoclasts
Paget’s disease is a late-onset disease that is quite common in the elderly Caucasian population, where it affects
approximately 3% of individuals (182). The disease is
characterized by focal increases in osteoclast numbers,
nuclearity, and size, which leads to localized bone destruction, although surrounding osteoblasts also are activated
(183) (Table 1). The identified causes of the disease include mutations in four different genes, TNFRSF11A,
TNFRSF11B, VCP, and SQSTM1 (182, 184 –186). These
genes encode RANK, OPG, p97, and p62, all of which are
involved in the regulation of osteoclastogenesis. The mutations all result in different subtypes of Paget’s (182,
184 –186). These mutations render the osteoclast precursors more sensitive to RANKL stimulation, resulting in a
higher number of osteoclasts, and potentially also explaining the presence of giant osteoclasts (185, 187, 188). Interestingly, a recent study in mice indicated that the most
common mutation in p62 does not make the osteoclasts
Pagetic alone, although it sensitizes them to other yet-tobe-described causes of Paget’s (189, 190).
In Paget’s patients, biochemical markers of both bone
resorption and bone formation are increased, showing an
overall increase in bone turnover at the affected sites.
However, bone resorption clearly exceeds bone formation
(182). Whether the osteoclasts in Paget’s behave differently from those in healthy individuals during bone resorption is presently unknown. In particular, it is not
known whether osteoclasts in Paget’s require acidification
to resorb bone, or whether cathepsin K is the main protease, although answers to these questions might be of
value in the development of new therapies for Paget’s.
Furthermore, an explanation for the localized nature of
Pagetic lesions has still not been found. Even under the
extreme circumstances seen in Pagetic lesions, bone formation is coupled to osteoclastic parameters, although
whether this is due to increased osteoclast numbers or
activities is not known. Moreover, in this case a treatment
type eliminating the activity of both types of cells is most
likely to be preferred because the increase in bone formation occurring is part of the pathology, and likely will
provide no benefit for the bones if maintained. Thus,
bisphosphonate, which strongly attenuated overall bone
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Henriksen et al.
Osteoclast Subtypes
turnover, appears to be highly relevant in the context of
Paget’s disease (191).
2. Osteolytic osteoclasts
Several forms of cancer can metastasize to bone and
form osteolytic metastases (192–196). Once the cancer
has reached the bone, tumor and bone interact in a vicious
cycle in which tumor-secreted factors, such as PTHrP,
stimulate bone cells, which in turn release growth factors
and cytokines that promote further tumor cell growth
(192, 197). The activation of osteoclastogenesis induced
by tumor cells has been shown to involve a switch in the
RANKL/OPG ratio favoring osteoclastogenesis and activation, leading to release of the tumorigenic factor TGF-␤,
and thereby inducing the vicious cycle (198). As a function
of the increased numbers of osteoclasts and accelerated
bone resorption, a marked up-regulation of osteoblast activities is also observed (199, 200).
The activity of osteoclasts in metastases has been monitored closely using biochemical markers of bone turnover
(199, 200), and these studies have indicated that bone
resorption by tumor-induced osteoclasts to some extent
depends on MMP activity, rather than cathepsin K, because the type I collagen fragment ICTP is released in high
amounts (199, 201) (Table 1). Animal models of breast
cancer bone metastases are to some extent sensitive to
inhibitors of both cathepsin K and MMPs (101, 202–204);
however, clinical data for MMP inhibitors have been disappointing (205, 206). An interesting question is whether
these agents, to be effective, have to inhibit MMPs before the
tumors actually metastasize. For cathepsin K inhibitors, the
data indicate a beneficial effect on the release of the bone
resorption marker N-terminal crosslinked peptide of type
I collagen, and an increase in ICTP levels (204). However,
further information is needed to draw reliable conclusions
on the usefulness of cathepsin K inhibitors for metastatic
bone disease.
In contrast, treatments ablating both osteoclasts and
the increased osteoblast activity, such as denosumab and
bisphosphonates, reduce the destructive capacity of the
metastasis and, importantly, the afflicted pain. However, they do not appear to affect the cancer cells (192,
207–210), although there are some indications that the
bisphosphonates affect the life span of the cancer cells
as well as reducing osteolysis (211).
In summary, from a treatment point of view, there are
several similarities between Paget’s and osteolytic metastases. The optimal approach appears to involve a strategy
of reducing overall bone turnover toward the normal
range, such as with the use of denosumab or bisphosphonates An intriguing possibility would be to target only the
areas undergoing destruction, but whether this is feasible
is presently not known.
Endocrine Reviews, February 2011, 32(1):31– 63
3. Arthritic osteoclasts
Later stages of RA are characterized by massive bone
destruction caused by osteoclasts (212, 213). However,
there are several indications that these osteoclasts are not
classical bone-resorbing osteoclasts but include cells that
degrade calcified cartilage (83, 214) (Table 1). Several
studies have indicated that TNF-␣ at least partially drives
osteoclastogenesis in RA (215), as exemplified by mice
overexpressing human TNF-␣ with massive joint destruction including bone erosion (216). Furthermore, TNF-␣neutralizing antibodies, such as infliximab, or soluble
TNF-␣ receptor antagonists, such as etanercept, provide
amelioration of RA in humans (217, 218). Apart from
TNF-␣, RANKL is, not surprisingly, a crucial factor in
osteoclastogenesis during RA (87), and mice deficient in
RANKL are protected against bone, but not cartilage, erosion (219). Treatment with OPG of mice with collageninduced arthritis also leads to amelioration of bone destruction, while having a markedly lower effect on
cartilage degradation (220). In addition, a study in which
TNF-␣ overexpressing mice were crossed with mice deficient in c-fos (i.e., deficient in osteoclasts), showed no bone
destruction but clear evidence of cartilage destruction
(221). Furthermore, human clinical trials using denosumab have demonstrated that the RANKL/RANK axis is
a key player in RA and that inhibition of RANKL signaling
may provide a useful treatment option (222). Finally,
more recent evidence has indicated that IL-1␣ and IL-1␤
both play a partial role in bone resorption and cartilage
degradation (223). Anakinra, which is a soluble IL-1
receptor antagonist, is also used for treatment of RA,
although it appears to be less effective than the TNF-␣
inhibitors (217). In addition, tocilizumab (anti-interleukin-6 receptor inhibitor) has shown promise in preventing RA progression through an effect including a
reduction in osteoclast numbers (158, 324).
Because osteoclasts play a significant role in RA,
bisphosphonates appear to be an attractive treatment option. Early evidence has indicated that zolendronate may
be useful (224), although this has not been fully established yet (225). Furthermore, interpretation of the effects
of bisphosphonates in RA is often clouded by glucocorticoid treatment of the same patients because glucocorticoids are associated with rapid systemic bone loss,
independent of RA (226, 227). However, in both collageninduced arthritis in rats and in the TNF-␣ transgenic
mouse model, zolendronate was effective in reducing both
bone and cartilage destruction (228, 229).
The bone resorption process in RA is still not completely understood despite several studies into the molecular mechanisms. The role of cathepsin K has been extensively studied, and the data are somewhat conflicting
Endocrine Reviews, February 2011, 32(1):31– 63
(230 –232). Overexpression of cathepsin K has been
shown to accelerate joint destruction in mice (231), and
overexpression of cathepsin K has been observed in humans with RA (233, 234). However, studies in the TNF-␣
overexpression model crossed with cathepsin K-deficient
mice showed that cathepsin K plays only a marginal role
in bone resorption in RA (232), a finding supported by a
case study showing severe arthritis in a pycnodysostotic
patient (235), although there are still controversies with
respect to the role of cathepsin K in RA (236).
Other cathepsins have not been explored in detail, and
their expression patterns do not indicate a particular effect
on osteoclast function in RA (237).
Under some circumstances, MMPs also play a role in
bone resorption (2, 81). Studies showing that the MMPderived collagen type I fragment, ICTP, is increased in RA
could indicate that osteoclasts used MMPs to digest matrix under these circumstances (238, 239). Infliximab
treatment has been shown to reduce ICTP levels, as well as
osteoclast numbers (240), further indicating that osteoclasts utilize MMP-mediated bone degradation in RA.
However, a direct link between the production of ICTP
and osteoclasts has not been demonstrated yet.
Whether acid secretion by osteoclasts is needed for
bone destruction in RA is also not clear. Because bone
destruction is likely to occur as a result of MMP activity,
the need for acidification may be reduced when compared
with “classical” bone resorption (69), although this is still
not fully understood. Another possibility is that MMPmediated collagen type I degradation is mediated by another cell type, although this still remains to be clarified.
A case study of arthritis in a case of autosomal dominant
osteopetrosis type II (241) showed a lack of bone degradation, whereas cartilage degradation was abundant,
thereby mimicking the situation seen in osteoclast-deficient systems (221) and indicating that bone resorption in
RA depends fully on acid secretion.
In summary, development of severe RA involves osteoclasts, and a reduction of bone resorption by these cells
is desired. This may be obtained through inhibition of
inflammation and thereby bone and cartilage destruction,
as seen with anti-TNF-␣ therapy. Alternatively, therapies
such as denosumab that target the osteoclasts directly may
also be useful, although these fail to eliminate inflammation and only partially prevent cartilage degradation
(220). The optimal therapy could be a combination of
antiinflammatory and antiosteoclastic measures, although this is presently not known.
In summary, studies of osteoclasts under pathological
circumstances have highlighted some important phenomena. First, osteoclasts themselves, not just their resorptive
activity, mediate bone formation and therefore perform an
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important secondary role in bone remodeling, which is of
importance when developing novel treatments for osteoporosis (15). Second, excessive and local activation of
osteoclasts occurs in several diseases, and interestingly
the osteoclasts appear to switch subtype with respect to
their resorption machinery. These findings highlight the
importance of characterizing the function of osteoclasts
under pathological circumstances to optimize treatment
strategies.
VI. Drug-Induced Osteoclast Subtypes
A. Existing drugs
Several antiresorptive drugs for the treatment of osteoporosis, as well as glucocorticoids and PTH treatment, are
known to alter osteoclasts in various ways. These drugs all
provide critical information on osteoclast function, and
furthermore, they have also played a great role in illustrating the interplay between osteoclasts and osteoblasts,
as will be described in the following section.
1. Bisphosphonates
Bisphosphonates have long been associated with induction of apoptosis in osteoclasts, and the mechanism of
action underlying the apoptotic effect depends on whether
or not the bisphosphonates contain nitrogen (242). Both
classes of bisphosphonates bind to the bone matrix and are
taken up by the osteoclast during bone resorption. The
simple bisphosphonates are metabolized into toxic ATP
analogs, thereby inducing osteoclast apoptosis in vitro
(242). The nitrogen-containing bisphosphonates exert
their function by inhibiting the mevalonate pathway,
which leads to the generation of an ATP analog known to
induce apoptosis in osteoclasts in vitro (242). The antiresorptive potency of the nitrogen-containing bisphosphonates in vivo is controlled by mineral binding affinity and
by their ability to inhibit the mevalonate pathway (242).
Although in vitro data clearly show that bisphosphonates induce apoptosis, analyses of osteoclast numbers in
iliac crest biopsies failed to show a reduction in the number
of osteoclasts when patients were treated with bisphosphonates (243–245). On the other hand, bisphosphonates
reduce systemic levels of TRACP 5b and cathepsin K, both
markers of osteoclast number (32, 246 –248), potentially
indicating that osteoclasts undergo systemic apoptosis,
which correlates well with the expected effects of bisphosphonates (242) (Table 1). Other studies have shown that
when bisphosphonate therapy continues for more than 1
yr, the number of circulating osteoclast precursors is reduced, and these reductions are speculated to be related to
reduced serum RANKL levels (249, 250).
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A recent study of biopsies from alendronate-treated patients showed the presence of giant hypernucleated, detached, and frequently apoptotic osteoclasts, and the number of these abnormal osteoclasts correlated with the
cumulative dose of bisphosphonate (251). Although interesting, the biological implications of this finding are not
clear yet.
One potential explanation for the discrepancies in scoring osteoclasts in the iliac crest biopsies is the very low
number of osteoclasts observed in general. Recent reports
have also debated the clinical relevance of studying iliac
crest biopsies because they are from non-weight-bearing
bones and these are different from weight-bearing bones
(252–254), and in general more data are needed to draw
a final conclusion on the osteoclastic response to
bisphosphonates.
On the other hand, the effect on reduction of bone resorption measured both by biochemical markers and by
bone histomorphometry (activation frequency) confirms a
potent reduction in bone resorption, and the level of
reduction is often down to the lower range of premenopausal levels, although this depends heavily on the efficacy of the individual bisphosphonate (242, 243, 245,
255–258).
With respect to secondary effects on bone formation,
measurement of biochemical markers of bone turnover
shows a marked reduction in bone formation markers,
and the effects are maintained throughout the treatment
period, although this again is dependent on the individual
bisphosphonate (242, 243, 245, 255–258). Biopsy studies
have confirmed that bone formation is reduced when compared with placebo, and although the reduction in bone
formation rates is dependent on the individual bisphosphonate, the data indicate that bone formation is not completely suppressed but is reduced to the lower postmenopausal levels (243, 245, 258). The FLEX study (Fracture
Intervention Trial Long-term Extension), although showing continued reductions in vertebral fractures, increase in
bone mineral density (BMD), and reduction of bone turnover markers with alendronate, did not show a significant
reduction in bone formation rates when comparing patients stopping alendronate to patients continuing treatment; however, the numbers of biopsies were low (259).
All in all, there is no doubt about the fracture-preventing effects of bisphosphonates; however, knowledge of the
effect of bisphosphonates on osteoclasts in vivo is quite
limited. Apoptosis of the osteoclasts most likely explains
the reduction in bone resorption. Furthermore, although
the extent of the secondary reduction in bone formation is
still discussed, it appears to be clinically relevant, and it
most likely is the explanation for the attenuation of the
BMD increase seen after the first year of treatment.
Endocrine Reviews, February 2011, 32(1):31– 63
a. Osteonecrosis of the jaw (ONJ) and bisphosphonates.
Bisphosphonate therapy, especially in the case of malignancy-induced bone loss, has been connected to the occurrence of ONJ, mainly due to the ability of bisphosphonates to strongly suppress bone turnover (260 –262).
Although the probability of ONJ is very low for the dosing
regimens used for treatment of osteoporosis, there has still
been a lot of debate about whether ONJ is the result of the
massive suppression of bone turnover in the jaw (262).
Interestingly, alveolar bone of the jaw is very similar to
bone matrix in the long bone, i.e., it contains the classical
cell types as well as the lamellar structure (263). Furthermore, bone remodeling occurs normally in alveolar bone,
although the rate of remodeling has been estimated to be
up to 10-fold higher than the corresponding rate in long
bones (263–266). In ONJ, the number of osteoclasts has
been investigated, and it appears that the osteoclasts are
absent from the lesions (267, 268), although opposing evidence also exists (269) and thus more studies are needed.
It has been speculated that massive suppression of osteoclast function, and thus bone turnover, in this highturnover compartment is what causes ONJ to occur; however, there are several other factors involved, such as tooth
extraction or infections, and the overall causality is still
not clear (262). One point of particular interest is
whether this phenomenon is specific for bisphosphonates or whether it will happen with other very potent
and long-lived antiresorptives; however, this is presently not known.
2. Selective estrogen receptor modulators/hormone
replacement therapy
Because cessation of estrogen production is a major
cause of osteoporosis (3, 14) and both estrogen and
SERMs are used for treatment of osteoporosis, several
studies have been conducted to clarify their effect on
osteoclasts.
Estrogen has been shown to exert direct antiosteoclastic effects at several stages of osteoclastic differentiation
and function, namely osteoclastogenesis, resorption, and
apoptosis. Direct inhibition of the formation of multinucleated osteoclasts is thought to be caused by suppression
of RANKL-induced c-Jun and basal c-Jun N-terminal kinase activity in osteoclast precursor cells (135, 136). In
nonpurified osteoclast-precursor systems, estrogen was
found to inhibit osteoclastic differentiation in a human
system (270), possibly via down-regulation of the ␣v␤3
integrin (271). Two studies of estrogen have been conducted using CD14⫹ osteoclast precursors. As mentioned
earlier, one study showed significant inhibition of osteoclastogenesis (137), whereas the other showed no direct
effect on osteoclast precursors (272). To date, there is no
explanation for this discrepancy.
Endocrine Reviews, February 2011, 32(1):31– 63
Studies of the effects of SERMs on osteoclasts have
shown that tamoxifen inhibits osteoclastogenesis directly,
whereas raloxifene and ospemifene only inhibited osteoclasts through up-regulation of the expression of OPG by
osteoblasts (273). Although early studies showed an effect
of raloxifene on osteoclastogenesis, these were conducted
using mixed cell populations and therefore most likely
reflect the increase in OPG (274).
Mature osteoclasts have also been shown to respond
directly to estrogen (275, 276). These studies showed that
both the activity and the production of the lysosomal enzymes are down-regulated by estrogen (277, 278), possibly explaining the reduction in resorption by the downregulation of cathepsin K and TRACP (36, 66, 77, 279)
(Table 1).
In summary, in vitro data clearly demonstrate that estrogen and SERMs reduce osteoclast numbers via inhibition of osteoclastogenesis, and potential effects on bone
resorption and apoptosis might add to the in vivo effect.
Although some studies of osteoclasts in patients treated
with either HRT or SERMs have been conducted, the effects of both estrogen and SERMs on bone remodeling
indices based on histomorphometry are quite modest
(280 –284). Overall, these studies show a reduction in activation, frequency, and depth of resorption, as well as—
where detectable—a small decrease in osteoclast numbers.
Reduced bone formation rates were also observed, confirming the coupled nature of inhibition mediated by estrogen and SERMs (280 –284). These data are corroborated by biochemical markers of bone turnover, which
clearly demonstrated a coupled reduction in bone resorption and bone formation (32, 285–287), and furthermore
explain the plateau effect observed in BMD measurements
after 1 yr (285).
In summary, many of the numerous studies of the in
vitro mode of action of HRT and SERMs show a reduction
in osteoclastogenesis. In alignment, in vivo studies of these
therapies on osteoclasts confirm that osteoclastogenesis is
lower than in the untreated population, and importantly,
these also confirm the secondary decrease in bone
formation.
3. Calcitonin
Calcitonin is a natural peptide hormone produced by
parafollicular cells (C cells) of the thyroid gland. Calcitonin possesses potent antiresorptive effects (288), and binding of calcitonin to the calcitonin receptor on osteoclasts
induces a rapid change in the cytoskeletal structure of the
osteoclasts in vitro, which in turn leads to a reduction in
bone resorption without inducing apoptosis of the cells
(102, 289, 290). Calcitonin in either an injectable or a
nasal form has been approved for treatment of osteoporosis; however, because it only prevents about 35% of
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vertebral fractures, most likely due to low exposure, the
clinical usefulness is limited (122). Recent studies have
indicated that a recently developed oral formulation of
salmon calcitonin will lead to improved efficacy because it
has been optimized with respect to pharmacokinetic and
pharmacodynamic properties. This has led to a 10-fold
higher exposure and thereby a greater reduction in bone
resorption parameters. Thus, this agent will most likely
provide improved efficacy in preventing fractures (291),
and although it remains to be proven in long-term clinical
trials, the phase II data are promising (127).
The mode of action of oral calcitonin is a transient
suppression of the nocturnal rise in bone resorption obtained by giving the treatment at the right time of day—in
the evening (292), which results in a reduction in bone
resorption, but no effect or very modest secondary effects
on bone formation (127) (Table 1). These findings are
further supported by other clinical studies showing that
calcitonin may inhibit bone resorption without affecting
bone formation, a finding observed independent of administration route (293–296).
There are histological indications that calcitonin attenuates ruffled border formation by osteoclasts (296 –298),
and this appears to be the mode of action underlying the
antiresorptive effects of calcitonin in vivo, thereby elaborating on the previously described transient reduction in
bone resorption (292).
Studies of mice lacking the calcitonin receptor indicated
that bone formation was increased, and thus that calcitonin is a suppressor of bone formation (299, 300). These
studies were conducted mainly in young mice. A recent
study in mice deficient in the calcitonin receptor specifically in osteoclasts failed to reproduce this finding (301,
302). However, considering the very modest, or nonexistent, suppression of bone formation in patients treated
with calcitonin, the mice data appear of low relevance in
the clinical setting (127, 293–296).
Further studies are needed to understand this potential
dissociation of bone resorption and bone formation. It
may be that this dissociation occurs because calcitonin
disappears quickly from the circulation and thus is a completely reversible treatment (122). An interesting question
is whether calcitonin treatment may result in better bone
quality than potent antiresorptives due to the lack of effect
on bone formation and the lower suppression of bone
resorption, which is expected to lead to a slow, yet prolonged increase in BMD (6, 303).
4. Parathyroid hormone
Although PTH does not appear to affect osteoclasts
directly because these cells do not appear to express the
PTH receptor, PTH nonetheless affects osteoclast function on many different levels (10). In vitro studies of the
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effects of PTH on osteoclasts all show that PTH induces
osteoclastogenesis and that induction of a transient
RANKL expression is essential for this effect (10). However, PTH has mainly been studied in relation to its powerful anabolic effects on osteoblasts (10). Intermittent dosing of PTH in human subjects results in a marked increase
in bone formation markers, and secondarily in activation
of bone resorption through increased RANKL expression
(9, 304, 305). Bone histomorphometric and biochemical
marker studies confirm the increase in bone turnover
(306 –308) (Table 1).
The anabolic mode of action of PTH has been debated
extensively. Studies show that PTH directly activates bone
formation by osteoblasts when given intermittently (309,
310). In mouse models that are either deficient in osteoclasts or deficient in bone resorption, data suggest that the
anabolic effect of PTH is dependent on the presence of
mature osteoclasts, but not on their activity (165, 166,
311). Furthermore, initial clinical trials combining alendronate and PTH showed that alendronate blunted the
anabolic effect of PTH (312, 313), and there were indications that even pretreatment with alendronate led to a
blunting of the PTH response (314). On the other hand,
animal studies indicate that PTH can be combined with a
bisphosphonate (315, 316), but, as noted by Johnston et
al. (315), there are marked differences in the doses of PTH
used in rodents and in humans.
Collectively, PTH exerts marked regulation of bone
turnover (15), including the activation of osteoclasts. The
potential anabolic role of osteoclasts and, especially, how
to achieve the right osteoclast subtype are debated intensely. Future studies will most likely explain this complex interplay between bone cells and thus guide the right
combination of PTH and antiresorptive.
5. Strontium ranelate
Strontium ranelate is approved for treatment of osteoporosis, albeit only in Europe, through its ability to reduce
fracture risk in patients (317–321). The mode of action has
been studied extensively, and yet it is not fully clear exactly
how it works in vivo. Bone biopsies have been investigated, and these indicated small increases in bone formation and mineralization rates but no changes in bone resorption or osteoclast parameters, thus indicating that
strontium ranelate stimulates novel bone formation (322).
These data were supported by analysis of biochemical
markers of bone turnover demonstrating increased bone
formation (308, 323), while also showing a modest decrease in bone resorption markers (323, 325).
In vitro studies support the hypothesis that strontium
ranelate has a dual effect, namely inhibition of bone resorption while stimulating bone formation (326 –329).
Endocrine Reviews, February 2011, 32(1):31– 63
Furthermore, strontium has also been shown to increase
OPG expression by osteoblasts (330).
In summary, strontium ranelate is a very interesting
molecule with respect to effects on osteoclasts, and several
lines of in vitro evidence indicate that it reduces osteoclast
function (326). However, the relevance of the effect on
osteoclasts is still debated, and thus the overall effects of
this “uncoupling” molecule are still not fully understood.
6. Glucocorticoids
Glucocorticoids are used to overcome inflammatory
conditions, such as inflammatory bowel diseases and RA
(331). Glucocorticoid use is associated with severe bone
loss due to strongly attenuated bone formation (332). This
attenuation of bone formation leads to a rapid acceleration in the number of fractures in glucocorticoid-treated
patients (332), especially in trabecular bone compartments such as vertebrae (333). Glucocorticoid treatment is
the most common cause of secondary osteoporosis (333),
and thus patients on glucocorticoids are often treated with
antiresorptives (334).
In vivo, glucocorticoids inhibit osteoblastogenesis, the
generation of bone-forming osteoblasts, and promote apoptosis of osteoblasts and osteocytes, which is consistent
with the well-known inhibition of bone formation (335).
In contrast, the cellular effects of glucocorticoids on
osteoclasts are a subject of controversy. In vivo, the effects
appear to fall into two categories, one being a short-lived
acceleration of osteoclastogenesis and bone resorption,
whereas the other is a reduction in osteoclast numbers,
which is not well-characterized with respect to exposure
time to glucocorticoids (335–340) (Table 1). Interestingly,
a study by Kim et al. (340) showed that the detrimental
effect of glucocorticoids on bone formation was absent
when the glucocorticoid receptor was ablated specifically
in osteoclasts in mice.
In vitro studies of glucocorticoids are often conducted
in the presence of contaminating cells, and because glucocorticoid treatment also promotes RANKL and reduces
OPG expression in osteoblasts, it is unclear exactly to
what extent they influence the osteoclasts (341, 342). Two
recent studies showed that glucocorticoid treatment hyperactivated osteoclasts and thus suggests that glucocorticoids indeed have a direct effect on bone resorption (343,
344). Yet some studies show the opposite (340). Overall,
the results appear to be very context-dependent, illustrating the complex nature of the biological effects of
glucocorticoids.
Measurements of biochemical markers of bone turnover in human subjects on glucocorticoid therapy provided diverse results, which appeared to be dependent on
the dose of glucocorticoid used (331, 345, 346). However,
biochemical marker data indicate that bone resorption
Endocrine Reviews, February 2011, 32(1):31– 63
increases short term, whereas bone formation is attenuated long term (345) in response to glucocorticoid therapy, which corresponds well to mouse studies (335–339).
The short-term increase in bone resorption and long-term
suppression of bone formation are also observed with histomorphometry in mice (347, 348).
In summary, glucocorticoids exert detrimental effects
on bone, and whereas the effects on osteoclasts are not
completely clear yet, further investigation of the effect on
the coupling between osteoclasts and osteoblasts could
explain the overall beneficial effect of antiresorptives on a
syndrome mediated primarily by suppressed bone formation (227, 349). These findings further highlight the importance of understanding the interplay between bone
cells to provide the optimal treatment.
B. Future treatments
A series of interesting targets for osteoporosis treatment are currently under investigation. The targets of
these treatments to some extent employ novel modes
of action on osteoclasts. These novel modes of action are
of importance when investigating whether they may have
secondary effects on bone formation, and subsequently on
bone quality.
1. Denosumab
Denosumab is a fully humanized monoclonal antibody
to RANKL; it has gone through a phase III fracture efficacy trial in which it was shown to reduce fracture rates by
68% in vertebrae and 40% in hip (351); and it was recently accepted for treatment of severe osteoporosis in
both the United States and Europe.
In line with in vitro studies of inhibition of RANKL
(210, 352), denosumab prevents osteoclastogenesis,
blocks bone resorption, and increases osteoclast apoptosis. It induces a massive reduction of osteoclasts in vivo
and, thereby, almost complete suppression of bone resorption in both humans and mice (352–354) (Table 1). Denosumab treatment also leads to a marked suppression of
bone formation markers in humans (353, 354), as well as
a marked suppression in bone formation rates measured
by histomorphometry in animal models (352, 355). Thus,
denosumab treatment is consistent with the classical perception of coupling.
A key point with respect to denosumab is whether the
suppression is too severe and could lead to detrimental
effects on bone quality long term (6). However, as is the
case with bisphosphonate treatment, this is not clear at
present.
2. Cathepsin K inhibitors
Cathepsin K is a critical protease for degradation of the
type I collagen matrix in the resorption pits during bone
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resorption by osteoclasts (36, 65, 66). Studies conducted
in pycnodysostosis patients before the final identification
of cathepsin K showed massive accumulation of nondigested bone collagen fibers in the resorption pit below the
osteoclasts (175). These findings were matched by those
from investigations in cathepsin K-deficient mice (66),
demonstrating a critical role for cathepsin K in degradation of the organic matrix. Further studies in cathepsin
K-deficient systems have indicated that cells of the osteoblast lineage, namely bone-lining cells (68); cells of hematopoietic origin (69); and a general up-regulation of the
osteoclastic stimuli, osteoclast numbers, and proteases,
especially RANKL and MMPs (103), are involved in compensating for the lack of cathepsin K. Interestingly, a hallmark of the absence of cathepsin K function is the presence
of the MMP-derived collagen fragment ICTP, which is
seen in pycnodysostosis patients, cathepsin K-deficient
mice, and cell cultures (67, 69, 103, 356), strongly indicating a compensation by MMPs in the absence of cathepsin K (Table 1).
An interesting study by Fuller et al. (357) showed that
inhibition of cathepsin K in cultured osteoclasts led to
augmented secretion of IGF-I. Furthermore, increased
numbers of osteoclasts, containing granules of matrix proteins, have been observed in monkey studies of cathepsin
K inhibitors (175, 181), thus indicating the potential of
this protease for anabolic stimulation of the osteoblasts.
Cathepsin K-deficient mice have been studied extensively,
and recent experiments indicate that bone formation in
trabecular bone is increased after cathepsin K administration and thus that bone resorption and bone formation are
not coupled (100, 176). However, clinical studies of cathepsin K inhibitors, such as odanacatib, have shown that
whereas a robust reduction in CTX and N-terminal
crosslinked peptide of type I collagen occurred and no
changes in TRACP 5b were observed, a significant decrease in the bone formation marker pro-peptide of collagen type I and nonsignificant reductions in bone formation rates by histomorphometry were seen (178).
Furthermore, a study of osteoclast morphology as a function of cathepsin K inhibition in humans indicated increased size of the osteoclasts and the presence of large
TRACP-positive vacuoles, yet no increase in osteoclast
numbers (358). Studies in monkeys clearly demonstrated
that bone formation in the trabecular compartments was
dose-dependent and significantly reduced by cathepsin K
inhibitors, whereas an induction of bone formation was
observed at cortical sites (179, 180). Further studies are
needed to clarify whether the osteoclasts in cathepsin Kdeficient situations indeed signal to the osteoblasts. An
indication came from a pycnodysostosis case study that
showed no bone formation response to PTH (174), and
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Osteoclast Subtypes
thus indicated that secretion of the coupling signals may be
attenuated at least in human systems. A possible explanation for the lack of secondary anabolic effects induced
by inhibition of cathepsin K is the presence of demineralized collagen fibers in the resorption pit, which are removed by bone-lining cells (68). Although it is not well
understood how the presence of fibers and their subsequent removal affect osteoblasts, a study indicated that
RGD sequences, which are numerous in collagen, antagonize osteoblast function (359).
These findings again illustrate the importance of carefully investigating the osteoclast subtype as a function of
cathepsin K inhibition to more accurately predict potential
secondary effects on bone formation.
3. Glucagon-like peptide-2
GLP-2 is a 33-amino acid peptide. GLP-2 is created by
specific posttranslational proteolytic cleavage of proglucagon in a process that also liberates the related GLP-1
(124). GLP-2 is produced by the intestinal endocrine L cell
and by various neurons in the central nervous system
(124). Intestinal GLP-2 is cosecreted along with GLP-1
upon nutrient ingestion.
GLP-2 has in clinical settings been demonstrated to inhibit bone resorption (124 –126) (Table 1). Reductions in
bone resorption by exogenous GLP-2 require an intact
gastrointestinal tract (125, 361, 362). The decreased mealinduced inhibition of bone resorption in jejunostomy patients, who lack a GLP-2 response, supports the view that
GLP-2 plays a role in postprandial reduction in bone resorption (361, 362).
GLP-2 has in addition been suggested to inhibit bone
resorption without affecting bone formation (125), highlighting this mode of inhibition of resorption for further
investigation with respect to osteoclast subtypes.
4. Acid secretion inhibitors
Acid secretion by osteoclasts has been an interesting
therapeutic target since the discovery that this process is
controlled by the a3 subunit of the V-ATPase and ClC-7,
both of which are quite specific to osteoclasts (37, 39, 56).
Furthermore, in vitro studies of osteoclasts treated with
inhibitors of these ion transporters have shown that the
osteoclasts are unable to resorb bone and that they therefore survive longer (8, 159, 363), thereby mimicking the
elevated numbers of osteoclasts observed in patients with
mutations in the genes for a3 and ClC-7 (37, 97) (Table 1).
In aged ovariectomized rats, early low-potency chloride
channel inhibitors were able to prevent bone resorption by
approximately 50%, as monitored by both BMD and the
biochemical markers of bone resorption CTX-I or deoxypyridinoline, while augmenting the number of osteoclasts
Endocrine Reviews, February 2011, 32(1):31– 63
and showing no inhibition of bone formation markers (8,
364). Similar findings were published for an inhibitor of
the V-ATPase (365). In a study of prosthetic implants
coated with bafilomycin, osteoclast numbers were elevated, and indications of increased bone formation were
observed (366). These studies were the first to provide
proof of concept that inhibition of acidification is a really
promising target for osteoporosis treatment. Most interestingly, bone formation levels, as measured by osteocalcin and by evaluation of the dynamic histomorphometry
parameters mineral apposition rate and the mineralizing
surface vs. bone surface, were not affected. These data
therefore suggest that inhibition of acidification of the
osteoclastic resorption lacunae results in an uncoupling of
bone formation and bone resorption, thereby possibly improving the potential efficacy of the treatment. This is in
contrast to other antiresorptive treatments where a secondary decrease in bone formation is observed (3, 367).
These data also indicate that the subtype of osteoclasts
obtained—nonresorbing yet alive—when targeting acid
secretion is active with respect to bone formation, and thus
might possibly be combined with PTH treatment in the
future.
Finally, other compounds that appear to modulate the
activity of osteoclasts are in development for osteoporosis.
These include calcilytics, PTHrP, and sclerostin, but their
effects on osteoclasts, which most likely are indirect, are
not clear yet (368 –370), and thus these will not be described further.
VII. The Bone Anabolic Effects of
the Osteoclasts
Since the early discovery that osteoclast activities were
involved in regulation of bone formation during targeted
remodeling (17, 18, 371, 372), a series of studies have
investigated the nature of this process.
The early studies focused mainly on the release of molecules from the bone matrix during bone resorption and
identified molecules such as IGF-I and TGF-␤ (357, 373,
374). However, with the recent discovery that mature osteoclasts, not osteoclast precursors and not necessarily
bone resorption, are needed for stimulation of bone formation (8, 9), a series of studies have investigated this
phenomenon.
Zhao et al. (12) demonstrated that osteoclast-mediated
expression of EphrinB2 and osteoblast-mediated expression of EphB4 were involved in a bidirectional communication between these cell types. EphrinB2 on osteoclasts
stimulated bone formation by the osteoblasts via binding
to EphB4, while EphB4 expression on osteoblasts in turn
inhibited osteoclastogenesis via binding to EphrinB2 (12).
Endocrine Reviews, February 2011, 32(1):31– 63
However, ephrin signaling requires close contact between
the osteoclasts and their target cells. This has led to the
speculation that ephrin signaling could be involved in the
interplay between osteoclasts and bone-lining cells, which
are found in close contact and appear to regulate the activity of each other (68, 375).
Stimulatory signals from osteoclasts directly to mature
bone-forming osteoblasts are, on the other hand, likely to
be paracrine because these cell types are not found in close
contact (264). Both TGF-␤ and IGF-I are produced by the
osteoclasts and are known to stimulate bone formation
under various circumstances (376 –380). In relation to
these findings, it is interesting that the anabolic effect of
PTH in mice was shown to be mediated through IGF-I
(350), an effect that is absent in the absence of osteoclasts
(166). This confirms that IGF-I is a coupling factor.
A recent study demonstrated the mature human osteoclasts, independent of their resorptive activity, secrete factors that activate nodule formation by the osteoblasts (11).
This study was followed by a study showing that osteoclasts produce the anabolic factors bone morphogenetic
protein 6, Wnt10b, and sphingosine-1-phosphate, again
independent of bone resorption (13). Furthermore, inhibition of bone morphogenetic protein 6, Wnt10b, and
sphingosine-1-phosphate led to inhibition of the osteoclast-mediated stimulation of bone formation in vitro.
Finally, osteoclasts have also been shown to produce cardiotrophin-1 (CT-1), which activates bone formation by
osteoblasts, although the role of CT-1 was clearly shown
to be dependent on age because loss of CT-1 in newborn
mice caused osteopenia, whereas in larger mice it caused
mild osteopetrosis due to defective bone resorption (360).
In summary, the presence of mature osteoclasts is associated with the secretion of stimulation of bone anabolic
signals, and whereas several candidate factors have been
identified, a clear demonstration that removal of one of the
molecules specifically in the osteoclasts in vivo leads to loss
of bone formation is still missing.
VIII. Conclusions and Future Perspectives
Osteoclasts have traditionally been viewed as bone resorption “machines”; however, studies of osteoclasts have
highlighted that these cells are highly context-specific, and
the context of the individual osteoclasts is important for
the continued regulation of bone remodeling.
As described in detail in this review, the osteoclasts
possess at least two highly important functions: 1) bone
resorption, a process that is highly dependent on a series
of external stimuli, such as matrix type, remodeling status,
hormones involved in calcium homeostasis, genotype, inflammation, and importantly also on intervention strate-
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gies; and 2) stimulation of bone formation by the osteoblasts, a process that as illustrated by studies conducted in
osteopetrotic patients is, to a large extent, independent of
bone resorption. It is presently not completely clear when
the osteoclasts are anabolically active, yet it appears to be
related to the presence of large multinuclear osteoclasts
because bone anabolic responses are seen under these circumstances (3, 15).
Understanding osteoclast functioning may be useful for
developing drugs that not only inhibit bone resorption but
also enable bone resorption levels that ensure targeted
remodeling and, importantly, support continued anabolic
signaling from osteoclasts to osteoblasts in the bone remodeling compartment. This has the triple effect of:
1) maintaining a sufficient resorption level and thereby
avoiding excessive aging of the bones; 2) sustaining a local
stimulation of bone formation at the resorption site only;
and 3) not initiating induction of bone formation in otherwise quiescent sites. Theoretically, this type of inhibition
of bone resorption would allow a continuous, ongoing
increase in BMD, which is in contrast to the effects of the
presently approved antiresorptives where a plateau effect
on BMD is observed within the first 12–18 months. This
means that even with less powerful suppression of bone
resorption, such as that seen with the oral formulation of
salmon calcitonin, the long-term effects would surpass
those of the bisphosphonates.
A deeper understanding of both the differences in the
resorption process depending on circumstances and the
knowledge relating to when the osteoclasts are anabolically active will aid in the identification of novel treatment
opportunities for bone diseases.
Finally, the use of biochemical markers of bone turnover is becoming increasingly relevant for the continued
understanding of osteoclasts. Markers provide systemic
information on the outcome of a given treatment and can
help answer questions such as whether glucocorticoids
exert detrimental effects on bone formation, and whether
antiresorptives antagonize bone formation secondary to
bone resorption because of suppression of osteoclast numbers or activity.
Acknowledgments
The Danish Research Foundation “Den Danske Forskningsfond” is acknowledged for financial support.
Address all correspondence and requests for reprints to: K. Henriksen,
Nordic Bioscience A/S, Herlev Hovedgade 207, DK-2730 Herlev, Denmark. E-mail: [email protected]
Disclosure Summary: M.A.K. owns stocks in Nordic Bioscience A/S.
All other authors have no conflicts of interest.
50
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Osteoclast Subtypes
Endocrine Reviews, February 2011, 32(1):31– 63
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Rev Endocr Metab Disord (2010) 11:219–227
DOI 10.1007/s11154-010-9153-1
Cellular mechanisms of bone remodeling
Erik Fink Eriksen
Published online: 29 December 2010
# The Author(s) 2010. This article is published with open access at Springerlink.com
Abstract Bone remodeling is a tightly regulated process
securing repair of microdamage (targeted remodeling) and
replacement of old bone with new bone through sequential
osteoclastic resorption and osteoblastic bone formation. The
rate of remodeling is regulated by a wide variety of
calcitropic hormones (PTH, thyroid hormone, sex steroids
etc.). In recent years we have come to appreciate that bone
remodeling proceeds in a specialized vascular structure,—
the Bone Remodeling Compartment (BRC). The outer
lining of this compartment is made up of flattened cells,
displaying all the characteristics of lining cells in bone
including expression of OPG and RANKL. Reduced bone
turnover leads to a decrease in the number of BRCs, while
increased turnover causes an increase in the number of
BRCs. The secretion of regulatory factors inside a confined
space separated from the bone marrow would facilitate
local regulation of the remodeling process without interference from growth factors secreted by blood cells in the
marrow space. The BRC also creates an environment where
cells inside the structure are exposed to denuded bone,
which may enable direct cellular interactions with integrins
and other matrix factors known to regulate osteoclast/
osteoblast activity. However, the denuded bone surface
inside the BRC also constitutes an ideal environment for
the seeding of bone metastases, known to have high affinity
for bone matrix. Circulating osteoclast- and osteoblast
precursor cells have been demonstrated in peripheral blood.
The dominant pathway regulating osteoclast recruitment is
the RANKL/OPG system, while many different factors
E. F. Eriksen (*)
Department Of Clinical Endocrinology, Oslo University Hospital,
Aker,
Trondheimsveien 235,
0514 Oslo, Norway
e-mail: [email protected]
(RUNX, Osterix) are involved in osteoblast differentiation.
Both pathways are modulated by calcitropic hormones.
Keywords Osteoblasts . Osteoclasts . Lining cells . Growth
factors . Cytokines . Bone remodeling . Osteoporosis . Bone
remodeling compartment
1 Introduction
Bone histomorphometry has given us great insights into
bone physiology and bone remodeling in particular.
Histomorphometric indices obtained using tetracycline
double labeling techniques are unique, because they
contrary to DXA and bone markers reflect cellular activity
of osteoclasts and osteoblasts using the incorporation of a
time marker, namely spaced administration of an agent
(tetracycline) reflecting ongoing active bone formation. The
study of bone remodeling originated with the classical
works of Harold Frost 40 years ago [1] and our ever
expanding understanding of this process is the basis for the
development of highly effective treatments for osteoporosis, that we have seen over the last 20 years.
2 The bone remodeling cycle
Although macroscopically the skeleton seems to be a static
organ, it is an extremely dynamic tissue at the microscopic
level. The ability of bone to sustain the tremendous loads
placed on it in everyday life depends on, constant repair of
mechanical microdamage that develops both in cancellous
bone—the “spongy” bone present in the vertebrae, pelvis, and
ends (metaphyses) of long bones—and in cortical bone—the
compact bone present in the shafts (diaphyses) of the long
bones and surrounding cancellous bone as a thin layer in the
220
Rev Endocr Metab Disord (2010) 11:219–227
vertebrae and pelvis. Bone remodeling is based on the
concerted action of resorptive and formative cell populations
in order to replace old bone with new bone and thus secure the
integrity of the skeleton. This sequence has to be tightly
regulated by both local and systemic factors, because
significant deviations from a neutral balance between resorption and formation would mean severe accelerated bone loss
or bone gains with possible disastrous consequences in terms
of increased fracture risk or compression syndromes.
Bone remodeling takes place in what Frost termed the
Basic Multicellular Unit (BMU), which comprises the
osteoclasts, osteoblasts, and osteocytes within the boneremodeling cavity (Fig. 1). In cancellous bone remodeling
occurs on the surface of trabeculae and lasts about 200 days
in normal bone. The remodeling cycle can be as short as
100 days in thyrotoxicosis and primary hyperparathyroidism and exceed 1,000 days in low turnover states like
Myxedema and after bisphosphonate treatment [2]. Remodeling is initiated by osteoclastic resorption, which erodes a
resorption lacuna, the depth of which varies between 60 in
young individuals and 40 μm in older individuals. The
resorption period has a median duration of 30–40 days and
is followed by bone formation over a period of 150 days
(Fig. 1) [3, 4]. In normal bone the result of the remodeling
cycle is complete refilling of the resorption lacuna with new
bone. In disease states like osteoporosis, the main defect is
that the osteoblast is unable to refill the resorption lacuna
leading to a net loss of bone with each remodeling event
Resorption
Formation
Marrow capillary
Cancellous bone
50 µm
80 µm
Cortical bone
Haversian system
Central vessel
0
100
200
Time -days
Fig. 1 Schematic representation of Bone Multicellular Units (BMUs)
in cancellous and cortical bone. Broken lines denote the outer limit of
Bone Remodeling Compartment associated with the resorptive and
formative sites of the BMU. The mean thickness of the structure in
cancellous bone is 50 μm and 80 μm in cortical bone equivalent to a
mean Haversian system diameter of 160 μm. The Blood supply for the
BRCs is provided by capillaries either coming from the marrow space
as is the case for cancellous BMUs or from the central vessel of
Haversian systems in cortical bone. The duration of the remodeling
sequence is somewhat longer in cancellous than in cortical bone. The
position of marrow cappillaries is hypothetical, as the exact
distribution is poorly elucidated
[5]. In cortical bone remodeling proceeds in tunnels with
osteoclasts forming “cutting cones” removing damaged
bone followed by refilling by osteoblasts in the “closing
cone” occurring behind the osteoclasts [6]. In normal bone
the duration of the remodeling cycle in cortical is shorter
than in cancellous bone with a median of 120 days [6]. The
total surface of cancellous bone is completely remodeled
over a period of 2 years.
Contrary to remodeling sites in cancellous bone,
which are close to red marrow, known to contain
osteoprogenitor cells [7], remodeling sites in cortical bone
are distant from red marrow. Therefore, it was assumed
that the mechanisms of bone remodeling were different in
cancellous versus cortical bone, i.e. that the cells needed
for bone remodeling in cancellous bone traveled directly
from the red marrow to bone surfaces in cancellous bone,
while cells reached cortical remodeling sites bone via the
vasculature [8].
2.1 Osteoblast differentiation
Osteoblasts are mesenchymal cells derived from mesodermal and neural crest progenitor cells and their formation
entails differentiation from progenitors into proliferating
preosteoblasts, bone matrix-producing osteoblasts, and
eventually into osteocytes or a bone-lining cells. The
earliest osteoblastic marker, Runt-related transcription
factor 2 (Runx2) is necessary for progenitor cell differentiation along the osteoblast lineage [9]. During this
sequence of cellular proliferation Runx2 regulates expression of genes encoding osteocalcin, VEGF, RANKL,
sclerostin, and dentin matrix protein 1 [DMP1] [10].
Osterix is another transcription factor essential for osteoblast differentiation [11]. A large number of paracrine,
autocrine, and endocrine factors affect osteoblast development and maturation like: bone morphogenetic proteins
(BMPs), growth factors like FGF and IGF, angiogenic
factors like endothelin-1, hormones like PTH and prostaglandin agonists, all modulate osteoblast differentiation
[12]. The action of PTH and BMPs is closely associated
with activation of Wnt signalling pathways [13].
The fully differentiated osteoblast is characterized by
coexpression of alkaline phosphatase and type I collagen,
both important for synthesis of bone matrix and
subsequent mineralization thereof [14]. Mature osteoblasts also produce regulators of matrix mineralization
like osteocalcin, osteopontin and ostenectin, RANKL
which is necessary for osteoclast differentiation as well
as the receptor for PTH (PTHR1). At the end of their
lifespan osteoblasts transform into either osteocytes which
become embedded in the mineralized matrix or lining
cells, which cover all surfaces of bone. Specific molecules
expressed by osteocytes include DMP1, FGF 23 and
Rev Endocr Metab Disord (2010) 11:219–227
sclerostin, which control bone formation and phosphate
metabolism [15].
2.1.1 Wnts and osteoblast differentiation
Wnts are secreted glycoproteins crucial for the development
and renewal of many tissues, including bone. Wnt
signalling dominate osteoblast differentiation pathways
and act via binding to a receptor complex consisting of
LDL receptor-related protein 5 (LRP5) orLRP6 and one of
ten Frizzled molecules [13]. The so called canonical Wnt
signaling pathway is active in all cells of the osteoblastic
lineage, and involves the stabilization of β-catenin and
regulation of multiple transcription factors [16]. Wnt/βcatenin signaling is also important for mechanotransduction, fracture healing and osteoclast maturation [17–19].
The activation of canonical Wnt-signaling promotes
osteoblast differentiation from mesenchymal progenitors at
the expense of adipogenesis, which leads to improved bone
strength, while suppression causes bone loss [20]. Canonical Wnt signaling in osteoblast differentiation is modulated
by Runx2 and osterix [21].
Wnt signaling is a prime target for bone active drugs and
the approaches include inhibition of Wnt antagonist like
Dkk1, sclerostin, and Sfrp1 with neutralizing antibodies
and inhibition of glycogen synthase kinase 3 β (GSK3 β),
which promotes phosphorylation and degradation of βcatenin. One of the most promising approaches so far has
been inhibition of the osteocyte protein sclerostin, which
exerts tonic inhibition of osteoblast activity [22]. Sclerostin
is the product of the SOST gene, which is mutated and
downregulated in patients with sclerosteosis and van
Buchem disease and sclerosteosis [23], which are diseases
characterized by high bone density. Expression levels of
sclerostin are repressed in response to mechanical loading and
intermittent PTH treatment [24]. Preliminary studies with a
humanized monoclonal antibody against sclerostin have
shown bone anabolism in animals as well as humans [25].
2.2 Osteoclast differentiation
The dominating pathway regulating osteoclast differentiation is the RANKL/RANK/OPG pathway. This pathway is
based on osteoblasts promoting osteoclast differentiation
through membrane presentation of RANKL and binding of
this factor to the membrane receptor RANK on mononuclear osteoclast precursors. Osteoclast differentiation is also
modulated by M-CSF [26]. The promotion of osteoclast
differentiation by RANKL is inhibited by the decoy
receptor osteoprotegerin (OPG), which is also produced
by osteoblasts [26] (74). Estrogens increase OPG and
decrease RANKL expression in osteblasts, thus favoring
bone formation. Postmenopausal bone loss is linked to
221
reduced estrogen levels favoring increased resorption. In
many instances [26]. PTH given as daily injections favors
bone anabolism, reduces RANKL and increases OPG
levels. In cases with chronic elevation of circulating PTH
levels as seen in primary hyperparathyroidism the opposite
pattern is seen with elevated RANKL and reduced OPG
levels. A humanized monoclonal antibody against RANKL
has been shown to elicit even more pronounced reduction
in osteoclast numbers [27] than bisphosphonates and has
demonstrated excellent reduction of fracture risk in postmenopausal osteoporosis [28].
2.3 Coupling between resorption and formation
During normal bone remodeling, the amount of resorbed
bone is completely replaced in location and amount by new
bone. This is secured through tight coupling of bone
resorption to bone formation. The mechanisms underlying
the coupling process still remains largely elusive, although
the last 15 years has increased our knowledge significantly.
The dominating hypothesis years ago was that liberation
of growth factors like IGF 1 and 2 and cytokines embedded
in bone matrix during bone resorption secured the balance
between resorption and formation during bone remodeling
[29]. Later work showing that osteoblastic bone formation
proceeds unperturbed despite lack of bone resorption in the
presence of defective osteoclasts lacking for example
chloride channels or important factors for ruffled border
formation in osteoclasts like c-Src [30] has supplemented
this hypothesis. The important role that osteoclasts play in
the regulation of bone formation is also corroborated by
studies on mice lacking c-fos or M-CSF, which display
absence of osteoclasts and defective bone formation [31].
Other system involved in coupling of bone resorption to
bone formation are the transmembrane proteins, ephrinB2,
which are expressed on osteoblasts and EPH receptor B4
(EphB4), which are expressed on osteoclasts [32]. Also the
osteoclastic factor sphingosine 1-phosphate (S1P) [33]
seems to play a significant role. The interaction of Ephrin
and EPH by cell to cell contact promotes osteoblast
differentiation and represses osteoclast differentiation.
Secretion of S1P by osteoclast seems to recruit osteoblast
progenitor cells to sites of bone resorption and stimulate
differentiation of these progenitor cells by stimulating
EphB4 signaling, This causes a shut down of bone
resorption and initiate the formative phase of bone
remodeling in the so called transition phase.
3 Targeted and non-targeted remodeling
Through its constant removal and renewal of damaged
bone, bone remodeling secures skeletal integrity throughout
222
life. It has become customary to distinguish between
targeted and non-targeted (stochastic) remodeling. Nontargeted remodeling denotes regulation remodeling by
hormones like PTH, thyroxine, growth hormone and
estrogen, but also antiresorptive drugs like bisphosphonates may affect non-targeted remodeling. It seems that
the main pathway is via modulation of osteoclasts, which
then via the coupling between resorption and formation
subsequently affects osteoblast activity. Targeted remodeling secures removal of damaged bone through targeted
resorption. Osteocytes are the most abundant cells in
bone, and their death by microdamage has been suggested to
be the major event leading in the initiation of osteoclastic bone
resorption. In normal bone [34]. Resorption lacunae are 3
times more frequent in association with microcracks, indicating
that remodeling is associated with repair of such microdamage
[35]. Damaged osteocytes promote differentiation of osteoclast precursors driven by secretion of M-CSF and RANKL
[36]. In cortical bone there is evidence to suggest, that
microdamage not only activates new BMUs, but may also
may direct the movement of existing BMUs as they tunnel
through the cortex. It also seems that the degree of damage to
the osteocyte network determines osteocyte metabolic
responses to loading and influences targeted remodeling [37].
Analysis of the relationship of between mean microcrack
length and BMU resorption space density in cortical bone
indicates that BMUs have an effective area about 40 times
greater than their actual cross-section, which suggests that
osteoclasts in the cutting cone of cortical BMUs are able to
sense and steer toward microdamage [38]. The relation
between microdamage and initiation of bone remodeling is
further corroborated by the fact that osteoclastic resorption
is augmented in old bone [39].
Rev Endocr Metab Disord (2010) 11:219–227
Table 1 Osteoblastic and endothelial markers detected on cells lining
the Bone remodeling Compartment (BRC) vs. vascular endothelial
cells as assessed by immuno- and enzyme histochemical staining
Antigen
BRC
Vascular endothelium
VEGF
Von Willebrand Factor
CD 34
Alkaline Phosphatase*
Osteocalcin
Osteonectin
IGF 1,2
TGF β 1,2,3
bFGF
OPG
RANKL
–
–
–
+
+
+
+
+
+
+
+
+
+
+
–
–
–
–
–
–
–
–
EG
EG
Ob
CV
OC
OC
4 The bone remodeling compartment
The work by Hauge et al. [40] demonstrated that the cells in
the BMU, even in cancellous bone, were not directly
contiguous to the bone marrow, but rather they were
covered by a “canopy” of cells forming the outer lining of
a specialized vascular structure with the denuded bone
surface as the other delineation. The cells of this canopy
display all classicial markers of the osteoblastic phenotype
(Table 1), and are therefore most probably bone-lining cells,
which seem to be connected to bone-lining cells on the
quiescent bone surface. The structure has been demonstrated in cortical as well as cortical bone (Fig. 2). In turn, these
bone-lining cells on the quiescent bone surface are in
communication with osteocytes embedded within the bone
matrix. Penetrating the canopy of bone-lining cells, and
presumably serving as a conduit for the cells needed in the
BMU, are capillaries.
Fig. 2 Different representations of BRC structures in cortical (upper
panel) and trabecular bone (lower panel). In cortical bone the BRC
(outer demarcation by the broken line) is filled with erythrocyte ghosts
(EG) and is located at the closing cone of the Haversian system
situated over osteoblasts (OB). A few osteoclasts (OC) are also seen.
CV denotes the central vessel of the Haversian system. In trabecular
bone (lower panel) the outer lining of the BRC is clearly discernible,
demarcating a vascular structure on top of osteoblasts (OB). Picture in
upper panel courtesy of Pierre Delmas, Lyon, France
Rev Endocr Metab Disord (2010) 11:219–227
Angiogenesis is closely associated with bone resorption
and bone and angiogenic factors like VEGF and endothelin
regulate osteoclast and osteoblast activity [41]. In addition
blood vessels serve as a way of transporting circulating
osteoblast [42] and osteoclast precursors [43] to sites
undergoing active remodeling. The involvement of vascular
cells during the initiation of bone resorption is still
unresolved. Is the very first step adhesion of a blood vessel
to bone lining cells at a site where targeted repair is
needed? Conceivably, osteocyte apoptosis and possible
release of osteotropic growth factors and cytokines could
be attractants for blood vessels, which would then subsequently initiate the formation of a resorptive BRC. But, as
outlined above, the framework for signaling within the
osteocyte-lining cell-BRC network could also be a way by
which remodeling events on bony surfaces are triggered
from damage accumulation or changes in mechanical strain
within bone.
There is increasing evidence for a common lineage and
close interaction between vascular endothelial cells and
bone cells. Endothelial cells drive differentiation of marrow
stromal cell towards the osteoblastic phenotype [44]
Endothelin and VEGF are also involved in signaling
between vasculature and bone [45], and VEGF as well as
other angiogenic factors are expressed during intramembranous osteogenesis. Osteoblastic cells, as well as osteoclasts, possess receptors for VEGF and also produce VEGF
[46]. Expression of VEGF is closely associated with the
early phases of bone modeling and remodeling events [47]
and it induces osteoblast chemotaxis and differentiation
[48] and differentiation.
Cells may enter the remodeling space either via
diapedesis through the lining cell dome covering the BRC
or via the circulation. It is still debatable whether all cells
involved in remodeling arrive via the circulation, but while
circulating osteoclast precursors were demonstrated more
than a decade ago, there is now increasing evidence that
osteoblast lineage cells are also present in the circulation
strengthening the involvement of circulating precursor cells
in the process [42, 49, 50].
While the systemic hormonal regulation of the remodeling process has to occur via factors arriving at individual
remodeling sites via the bloodstream, the way by which
local regulatory factors exert their action on individual cell
populations involved is still obscure. Over the last decades,
however, we have increased our knowledge about the
different growth factors and cytokines involved in local
regulation of bone remodeling tremendously (Fig. 3). Apart
from growth factors and cytokines, simple molecules like
nitrogen oxide (NO), as well as hypoxia and acidosis have
been shown to exert pronounced effects on bone remodeling balance and activity. NO exerts biphasic effects on
osteoclast activity with low concentrations potentiating and
223
high concentrations inhibiting bone resorption [51]. Similarly, osteoblastic growth and differentiation are inhibited
by high concentrations of NO, while lower concentrations
may play a role in regulating normal osteoblast growth and
in mediating the effects of estrogens on bone formation,
mechanotransduction and bone anabolic responses [51].
The dominating isoform of nitrogen oxide synthase (eNOS)
is expressed in osteocytes and lining cells, but not in
cuboidal osteoblasts [52]. Acidosis and hypoxia generally
increase bone resorption [53–56] and inhibit bone formation [57]. As hypoxia may cause acidosis through increased
anaerobic metabolism, the two factors may act synergistically at the tissue level [56]. Hypoxia and acidosis also
affect secretion of pro-angiogenic factors like VEGF as
outlined below.
5 Key functions of the bone remodeling compartment
1. The BRC provides a closed microenvironment permitting tight regulation of bone remodeling. Current
concepts regarding local regulation of bone remodeling
generally assumes that the local growth factors,
cytokines and even NO come either from cells in the
marrow space or vascular cells having free access to the
remodeling site without barriers, or are produced by
osteoclasts and osteoblasts at the remodeling site. The
BRC concept implies that the all factors liberated from
the cells or vessels in the marrow space exert their
regulatory role either through diffusion through the
outer layer of the BRC, transport via the bloodstream to
the interior of the BRC or indirectly via modulation of
cell activity in the outer wall of the BRC. The presence
of a specific compartment in which remodeling can
proceed without interference from local factors liberated in the marrow space seems to be logical. If the
access to the marrow space was open, the very high
levels of growth factors in the marrow microenvironment might offset eventual localized regulatory effects
by local growth factors, crucial to osteoclast and
osteoblast differentiation and the remodeling process.
2. The BRC is the structure translating microdamage into
targeted remodeling by which mechanosensory signals
from the osteocyte network are translated into changes
in osteoclast and osteoblast activity on trabecular
surfaces. Lining cells are connected to the osteocyte
network via gap junctions between lining cells on
quiescent surfaces and osteocyte cannaliculi [58]
(Fig. 4). Signals from lining cells indicating damage
or stress could be transmitted to the outer lining cell
layer of the BRC and trigger osteoclast recruitment. By
analogy with remodeling in cortical bone, which is
clearly associated with growth of a blood vessel into
224
Rev Endocr Metab Disord (2010) 11:219–227
MONOCYTES
VEGF, FGF,
ENDOTHELIN,N
O, H+, HYPOXIA
IL1,6,7,11, TNFα
Marrow capillary/sinusoids
E2, PTH, 1,25D,T3
IGF-2, IGF-1,
TGFβ,
β
-
-
OB
OC
IL,M-CSF. C-fos TNF,
EphrinB2
Osteoblastic
Lining cells
Vascular endothelium
EphB4
RANKL
EphrinB2
Osteoclast
precursor
EphB4
RANK reseptor
IGFs, S1P
Osteoblast
Osteoclast
Monocyte
Osteoclast
Fig. 3 Depiction of some of the main local regulatory factors
operating at remodeling sites with osteoclasts (OC) and osteoblasts
(OB). Interleukins (IL), tumor necrosis factors (TNF), transforming
growth factors (TGF), colony stimulating factors (CSF), Insulin like
growth factors (IGF), fibroblast growth factors (FGF), platelet derived
growth factors (PDGF), bone morphogenetic proteins (BMP)) are
formed by both monocytic cells in the marrow space or circulation, as
well as bone cells in the BMU. NFκB- or RANK- ligand (RANKL)
and osteoprotegerin (OPG) are formed specifically by osteoblasts.
Factors from the marrow space as well as factors liberated by
MARROW CAPILLARY
OC
OB
OSTEOCYTES
Fig. 4 Connections between the osteocyte network, lining cells and the
BRC. All cells in this network are connected with gap junctions, which
may provide a pathway (block arrows), by which signals generated deep
within bone may reach the surface and elicit remodeling events by
osteoclasts (OC) and osteoblasts (OB) in response to mechanical
stimuli. The response may be modulated by factors liberated from the
vascular endothelium or marrow capillaries/sinusoids and paracrine
factors (broken arrow) liberated from lining cells may also play a role
endothelial cells (vascular endothelial growth factor (VEGF), endothelin, nitrogen oxide (NO)) may diffuse to receptors on osteoclasts or
osteoblasts. The cellular responses in the BMU are then further
modulated by systemic hormones in the circulation (estrogen (E2),
parathyroid hormone (PTH), active vitamin D (1,25D), thyroid
hormone (T3)). Left lower insert depicts in detail osteoblastosteoclast interactions inside the BRC and right lower insert depict
an alternativ, still hypohetical, version of that interaction based on
lining cells acting as the osteoblastic component in thata interaction
the remodeling site (5), the presumed ingrowth of a
capillary into the BRC provides the vascular supply for
the cells in the BMU of cancellous bone and might also
provide the necessary osteoclasts and, subsequently, the
osteoblasts that are needed for bone remodeling in both
cancellous and cortical bone The BRC would also be a
site where hormonal modulation (e.g. ERT) of the
mechanosensory input could take place [59].
3. The BRC is the most probable structure at which
coupling between osteoclasts and osteoblasts occurs.
The RANKL/OPG pathway involves presentation of
osteoblastic, membrane bound RANKL to the RANK
receptor on osteoclast precursors by cell to cell contact.
Due to the timing and sequence of bone resorption and
bone formation, however, resorption and formation are
generally separated in time and space, which makes the
needed cell to cell contact between osteoclast precursors and active osteoblasts highly unlikely on a broader
basis, and even if soluble RANKL played a major role
it had no RANKL on precursor cells within the BRC to
Rev Endocr Metab Disord (2010) 11:219–227
bind to. A more likely cell, which could present RANKL
to RANK on osteoclast precursors, would be the lining
cell. As demonstrated in animals [60] and in humans
[61], lining cells exhibit positive immunoreactivity for
OPG and RANKL, and might therefore be responsible
for the cell to cell contact with osteoclast precursors.
4. The BRC also obviates the need for a “postal code”
system ensuring that resorptive and formative cells
adhere to areas on the bone surface, where they are
needed. Bone surfaces are generally covered by lining
cells, which would prevent direct contact between bone
cells and integrins or other adhesion molecules known
to modulate cell activity. The BRC would be the only
place where these cells (circulating osteoclasts as well
as circulating osteoblast precursors) would be exposed
to these matrix constituents, because the formation of
the BRC involves detachment of lining cells from the
bone surface.
5. The BRC may play a crucial role in the spread of bone
metastases. It is well established that apart from
entering bone via local ingrowth, tumor cells reach
bony surfaces via the circulation. The growth of
metastatic cells in bone is enhanced by the so called
“vicious cycle”, where PTHrp produced by tumor cells
(e.g. breast cancer cells) induces increased local bone
resorption and subsequent liberation of TGFβ from the
bone matrix [62]. The local effects of TGFβ in the
bone microenvironment are two-fold: 1) it enhances the
growth of bone metastases and 2) increases PTHrp
formation from tumor cells further [62], thus maintaining the vicious cycle. As shown above, one of the key
components of the vicious cycle, TGFβ, is produced by
the cells lining the BRC. Other key promoters of bone
metastases like IL-1 and IL-6 are also produced by the
lining cell layer covering BRCs. It is therefore probable
that the microenvironment in the BRC is highly
conducive to metastatic seeding and the formation of
the vicious cycle, further enabling growth of the bone
metastasis. Moreover, the existence of a closed compartment would make vicious cycle formation easier
due to absence of interference with cytokine and
growth factors from the marrow space. Several large
scale studies have established that bisphosphonates
reduce the number of skeletal events in breast cancer,
prostate cancer and myelomatosis, and iv bisphosphonates are now used routinely in advanced cancer [63].
There is still debate as to how much of the beneficial
effects of bisphosphonates in advanced cancer are due
to inhibition of angiogenesis or to other, direct antitumor effects. Bisphosphonate, however, could exert
their inhibitory effects on bone metastases simply by
reducing the number of BRCs and thereby the surface
of denuded bone available for metastatic seeding.
225
6 Conclusion
Bone remodeling involves tight coupling and regulation of
osteoclasts and osteoblasts and is modulated by a wide variety
of hormones and osteocyte products secreted in response to
mechanical stimulation and microdamage. Bone remodeling
proceeds in a specialized vascular entity the “Bone Remodeling Compartment” (BRC), which provides the structural basis
for coupling and regulation of cellular activity. Increased
knowledge about the interplay between different factors and
cells surrounding the BRC will most likely result in even
better treatment options for skeletal disease.
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
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