Vol. 17, No. 4
Printed in U.S.A.
0163-769X/96/$03.00/0
Endocrine Reviews
Copyright © 1996 by The Endocrine Society
Advances in Bone Biology: The Osteoclast'
G. DAVID ROODMAN
Department of Medicine, Division of Hematology, University of Texas Health Science Center, and the
Audie L. Murphy Veterans Administration Hospital, Research Service, San Antonio, Texas 78284-7880
XV. Summary and Conclusions
I. Introduction
II. Osteoclast Morphology
III. Phenotypic Characteristics of the Osteoclast
A. Marker enzymes for osteoclasts
B. Surface phenotype of the osteoclast
C. Calcitonin receptor
D. PTH receptor
E. pp60c-src
IV. Origin of the Osteoclast
A. Hematopoietic origin of the osteoclast
B. Osteoclast precursors
C. Characteristics of osteoclast precursors
V. Model Systems for Studying Osteoclast Formation and
Bone Resorption
A. Isolation of mature osteoclasts from bone
B. Giant cell tumors as a model of osteoclasts
C. In vivo models of osteoclast formation
D. Bone organ culture systems
E. Bone marrow culture systems
F. Osteoclast-like cell lines
VI. Factors That Affect Osteoclast Function and Formation
A. Systemic hormones
B. Factors that enhance osteoclast activity
C Inhibitory factors
VII. Fusion Factors Involved in Osteoclast Formation
VIII. Osteoclast Adhesion Molecules
IX. The Role of the Bone Microenvironment in Osteoclast
Formation
X. The Osteoclast as a Secretory Cell
XI. Protooncogenes Involved in Osteoclast Differentiation
and Bone Resorption
I. Introduction
B
ONE is a dynamic tissue that constantly undergoes remodeling. Bone remodeling is a coupled process in
which bone resorption is normally followed by new bone
formation. During early life, bone formation exceeds bone
resorption with a net increase in bone mass, while late in life,
bone resorption exceeds bone formation with net loss of
bone. During some pathological processes, such as in patients with advanced stages of multiple myeloma, bone remodeling is uncoupled, and bone resorption is not followed
by new bone formation. The primary cell responsible for
bone resorption is the multinucleated osteoclast. Although
many questions still remain unanswered about the factors
that regulate osteoclast formation and osteoclastic bone resorption, major advances have been made recently in our
understanding of the cell biology and molecular biology of
the osteoclast, as well as the role that the marrow microenvironment plays in regulating osteoclast formation and bone
resorption. The development of in vitro and in vivo model
systems over the last 10 yr has provided important new
information on the biology of the osteoclast in both normal
and pathological conditions.
II. Osteoclast Morphology
The osteoclast is a large multinucleated giant cell that
contains between 2 and 100 nuclei per cell but usually contains between 10 and 20 nuclei per cell (1) and differs from
macrophage polykaryons (Table 1). It can range in size up to
100 jam in diameter and is located on endosteal surfaces
within the Haversian system and on the periosteal surface
beneath the periosteum. Osteoclasts are usually extremely
rare cells in bone with only two to three per /xm3 (2). Osteoclast numbers are increased at sites of active bone turnover,
such as in the metaphysis of growing bone. Osteoclasts are
usually seen attached to the bone surface and are only rarely
seen away from the surface of bone. A characteristic feature
of osteoclasts that is not seen with macrophage polykaryons
is the ruffled border, which can be visualized by light microscopy but can be observed more easily by electron microscopy (1). The ruffled border is comprised of a series of
fine finger-like cytoplasmic projections of the plasma membrane adjacent to bone. Resorption and degradation of mineralized bone matrix occur beneath the ruffled border due to
the release of proteolytic enzymes and hydrogen ions across
A. c-fos
B. pp60c-src
XII. Mechanisms of Osteoclastic Bone Resorption
XIII. Osteoclast Apoptosis
XIV. Abnormalities in Osteoclast Function
A. Paget's disease
B. Hypercalcemia of malignancy
C. Osteopetrosis
D. Osteoporosis
Address reprint requests to: G. David Roodman, M.D., Ph.D., Research Service (151), Audie Murphy Veterans Administration Hospital,
7400 Merton Minter Boulevard, San Antonio, Texas 78284.
* Supported by Research Funds from the Veterans Administration
and Grant AM-35188 from the National Institutes of Diabetes and Digestive and Kidney Disease; Grant CA-40035 from the National Cancer
Institute; Grant AG-13652 from the National Institute on Aging; and
Grants AR-39529 and AR-41336 from the National Institute of Arthritis
and Musculoskeletal and Skin Diseases.
308
August, 1996
THE OSTEOCLAST
TABLE 1. Functional and phenotypic differences between
osteoclasts and macrophage polykaryons
Osteoclasts
Macrophage
polykaryons
Formation of resorption lacunae
Presence of a ruffled border
Expression of vitronectin receptor
Expression of calcitonin receptors
Contraction and immobilization in
response to calcitonin
Expression of TRAP in vivo
Reactivity of the 12 IF antibody
Expression of Fc receptors
Expression of nonspecific esterase
The presence of phenotypic markers on the cell is denoted by (+);
the absence of phenotypic markers is denoted by (—). Low abundance
of phenotypic markers is denoted by (±).
s
s
the ruffled border into the sealing zone (3). The plasma
membrane of the osteoclast is closely apposed to the bone
surface with an adjacent "clear zone," which is an organellefree area that is rich in actin-like filaments. Other ultrastructural characteristics of the osteoclast include large numbers
of lysosomes, numerous and pleomorphic mitochondria, and
extensive Golgi complexes located in the perinuclear area (3).
The cytoplasm contains dense core granules, and the nuclei
are usually centrally located but have a highly variable shape
and usually one or two prominent nucleoli per nucleus (1).
The rough endoplasmic reticulum is usually sparse, and free
ribosomes are numerous and can occur as single ribosomes
or as polyribosomes.
Scanning electron microscopy studies have shown the convoluted plasma membrane and the many long cytoplasmic
processes of osteoclasts when they attach to bone (4). On
scanning electron microscopy, osteoclasts have prominent
microvilli over the central portion of the cells, and when
osteoclasts are plated on bone surfaces, a characteristic resorption pit is formed below the cell at the site of attachment
of the ruffled border (5). These resorption lacunae are never
seen in the absence of osteoclasts and are not produced by
macrophages or macrophage polykaryons (6). Time-lapse
cinemicrography studies have demonstrated that osteoclasts
are highly motile cells, which become contracted when exposed to calcitonin or prostaglandin E2 (7). Immobilization of
osteoclasts by calcitonin is a unique feature of mammalian
osteoclasts and is not seen when macrophage polykaryons
are treated with calcitonin (7). Thus, osteoclasts have several
distinctive morphological characteristics that help distinguish them from other multinucleated giant cells such as
macrophage polykaryons or megakaryocytes.
III. Phenotypic Characteristics of the Osteoclast
A. Marker enzymes for osteoclasts
In addition to morphological differences, osteoclasts also
express several unique phenotypic features that distinguish
them from other multinucleated giant cells (Table 1). The
osteoclast contains high levels of acid hydrolases, as detected
by immunohistochemical studies (8). Tartrate-resistant acid
phosphatase (TRAP) (purple acid phosphatase, type 5 acid
phosphatase) is expressed at high levels in osteoclasts and is
309
present in lysosomes, Golgi, extracellular channels of the
ruffled border, and the space between the cells and bone (8).
Until recently, the tissue distribution of TRAP was thought
to be relatively restricted to the osteoclast in normal subjects
and to hairy cell leukemia cells or splenic macrophages in
patients with Gaucher's disease. However, more recent studies, using RT-PCR techniques, have shown that TRAP is
expressed in a variety of tissues including the gut, kidney,
and lung (9). Bone appears to express the highest levels of
TRAP among normal tissues, and TRAP can serve as a
marker enzyme for osteoclasts in bone. Murine and human
TRAP have been cloned, and the promoters for these genes
characterized (9,10). The 5'-flanking region of the TRAP gene
contains two promoters, one of which is within an intron, as
well as positive and negative regulatory elements. The TRAP
promoter has been used for targeted gene expression in the
osteoclast in transgenic mice (11). The physiological function
of TRAP in the osteoclast is still unclear. Studies by Zaidi et
al. (12), using a blocking antibody to TRAP, suggested that
TRAP may play an important role in osteoclastic bone resorption. However, recent studies using the techniques of
homologous recombination to create a nonfunctional TRAP
gene in mice, have demonstrated that "knocking out" the
TRAP gene resulted in developmental abnormalities in the
appendicular and axial skeleton, suggesting a role for TRAP
in endochondral bone formation (13). Importantly, these animals did not develop osteopetrosis. These data suggest that
TRAP is not involved primarily in osteoclastic bone resorption, but as a tyrosine phosphatase, must have other important functions that are not as yet delineated.
Minkin and co-workers (14) have used RT-PCR to distinguish authentic osteoclasts from splenic macrophage
polykaryons. These workers have detected osteopontin
mRNA in osteoclasts, as well as carbonic anhydrase II, the
calcitonin receptor, and confirmed that mRNAs associated
with the osteoblast phenotype, such as alkaline phosphatase
and osteocalcin, were absent in the osteoclast. Levels of carbonic anhydrase II are elevated in osteoclasts (15), and carbonic anhydrase II appears to play an important role in
osteoclastic bone resorption. Antisense constructs to carbonic anhydrase II block osteoclastic bone resorption both in
isolated osteoclasts and in bone organ cultures (16). Similarly, Asotra and colleagues (17) have shown that rabbit
osteoclasts that are actively resorbing bone express higher
levels of the carbonic anhydrase II mRNA than resting osteoclasts, as do human osteoclast-like cells isolated from
giant cell tumors of bone (18). These data demonstrate that
RT-PCR approaches can provide important information on
osteoclast gene expression and should be useful in examining changes in gene expression during osteoclast differentiation and activation. These techniques have already been
applied to studies of calcitonin receptor expression during
osteoclast differentiation (see below).
Recently, a novel human cysteine protease, termed cathepsin K or cathepsin O, has been cloned; this protease is
similar to an enzyme cloned from a rabbit osteoclast cDNA
library by Inaoka and co-workers (19) and may play an
important role in the bone-resorptive process. This protease
is predominantly expressed in osteoclasts, and the cDNA
encodes a protein of 329 amino acid residues, homologous to
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ROODMAN
the rabbit enzyme. High levels of cathepsin K are present in
osteoclasts from osteoarthritic hips and in giant cells from
giant cell tumors of bone, suggesting that it has a role in the
bone resorptive process. Expression of cathepsin K is not
unique to osteoclasts, since it has also been cloned from a
monocyte-derived macrophage cDNA library by Shi and
co-workers (20). Cathepsin K has potent endoprotease activity at acid pH and may play an important role in extracellular matrix degradation by osteoclasts. In addition to
high levels of cathepsin K, osteoclasts also contain lower
levels of cathepsin B, D, and L. Cathepsin B and L are rapidly
released into the extracellular matrix and participate in the
degradation of organic bone matrix near the ruffled border
during osteoclastic bone resorption (21).
Another marker enzyme for the osteoclast appears to be
matrix metalloproteinase 9 (MMP-9), also known as 92 kDa
gelatinase or type IV collagenase (22). MMP-9 can cleave the
a2- chain of type I collagen and collagen types III, IV, and V,
as well as gelatins. Several studies have demonstrated that
MMP-9 is localized exclusively in osteoclasts in bone tissues
from normal subjects and patients with rheumatoid arthritis
or metastatic carcinoma. Okada and co-workers (22) have
also shown that the MMP-9 protein is produced by osteoclasts in human bone tissues and have suggested that it
degrades bone collagen proteins below the ruffled border in
concert with MMP-1 and cysteine proteases. Similarly,
Wucherpfennig et al. (23) have also reported that giant cells
from human osteoclastomas express high levels of MMP-9,
as do osteoclasts from patients with Pa get's disease. Differential screening of a human osteoclastoma cDNA library by
these investigators found a large number of cDNA clones for
MMP-9. These data demonstrate that high levels of MMP-9
are expressed in osteoclasts and suggest an important role for
this enzyme in the degradation of collagen in the resorption
lacunae during bone remodeling. Consistent with these observations are the studies of Hill et al. (24) who reported that
selective inhibitors of gelatinase A and B and collagenase
inhibited interleukin-1 (IL-l)-stimulated bone resorption by
isolated rat osteoclasts. In contrast, Fuller and Chambers (25)
found that inhibitors of collagenase did not inhibit osteoclastic bone resorption, and in situ hybridization studies did not
demonstrate expression of the mRNA for collagenase in rat
osteoclasts. Thus, although the majority of studies demonstrate MMP-9 in osteoclasts, it is unclear whether osteoclasts
express collagenase. Most probably, collagenase produced
by osteoblasts is activated by osteoclasts during bone resorption and works with MMP-9 to degrade bone collagen
during the resorptive process. In preliminary studies, we
have shown that MMP-9 is expressed early during human
osteoclast differentiation even before TRAP (Fig. 1). Thus, the
osteoclast contains high levels of several enzymes that distinguish it from other bone cells. These include TRAP,
MMP-9, carbonic anhydrase II, and cathepsin O.
B. Surface phenotype of the osteoclast
The surface phenotype of osteoclasts has been described in
detail by Athanasou and co-workers (26). They have shown
that human, rabbit, and avian osteoclasts react with monoclonal antibodies against human ^-integrins, as well as the
Expression of Phenotypic Markers During
Osteoclast Differentation
CFU-GM
Early
Precursor
Committed
Precursor
Mature OCL
MMP-9
TRAP
VNR
CTR
FIG. 1. Expression of phenotypic markers during osteoclast generation. In this model of osteoclast differentiation, MMP-9 is the earliest
phenotypic marker expressed by cells in the osteoclast lineage. As the
cells differentiate, they begin expressing TRAP, followed by the vitronectin receptor. When the cells are committed to the osteoclast
lineage, the calcitonin receptor is expressed. MMP-9, TRAP, the vitronectin receptor, and the calcitonin receptor are all expressed in
mature osteoclasts.
human macrophage-associated CD68 antigen (27). In addition, avian osteoclasts also react with CDlla/18 and CD14,
which are not present on human osteoclasts. Osteoclasts lack
Fc receptors in contrast to macrophage polykaryons (26).
Using monoclonal antibody techniques, workers have attempted to identify unique surface antigens on osteoclasts,
but to date, most investigators have identified antigens that
are preferentially, but not exclusively, expressed on osteoclasts. Oursler and co-workers (28) were the first to develop
a monoclonal antibody, which they termed 121F, that initially appeared to be restricted to chicken osteoclasts. The
121F antibody identifies a novel plasma membrane glycoprotein that is expressed during the course of osteoclast
differentiation and shares structural and functional homology with the magnesium iron superoxide dismutase (29).
These authors have proposed that the 121F antigen protects
the osteoclast from the damaging effects of superoxide radicals generated during active bone resorption by converting
these toxic oxygen radicals at the cell surface to H202. Expression of the 121F antigen also can be induced in avian
bone marrow-derived multinucleated giant cells by treating
the cells with osteoblast-conditioned media or phorbol esters
in vitro (30). The 121F antibody also appears to react with
human osteoclasts and does not react with macrophage
polykaryons (30, 31).
Horton and co-workers (32) also have developed monoclonal antibodies that react with the osteoclast. These investigators used osteoclastoma tissue from human giant cell
tumors of bone as their immunogen. These authors developed several antibodies including 23c6 and 13c2, which react
with the osteoclast vitronectin receptor, a member of the
integrin family of adhesive molecules. Initially, the 23c6 antibody was thought to be specific for osteoclasts (32), but
others have recently reported that macrophages and macrophage polykaryons express the vitronectin receptor (26,
33). Kukita and co-workers (34) have shown that the 23c6
monoclonal antibody reacts with about one half of multinucleated cells formed in human marrow cultures, and several
different laboratories have used this antibody to purify os-
THE OSTEOCLAST
August, 1996
teoclast-like cells from giant cell tumors of bone to a high
degree of purity (31, 35).
Kukita and co-workers (36) have also developed a monoclonal antibody Kn22 that reacts with osteoclasts and osteoclast precursors (Fig. 2). This antibody can also react with
cells early in the macrophage lineage, and, in addition, has
the capacity to enrich osteoclast precursors. The antigen for
the Kn22 antibody is a 50-kDa protein that is expressed on
the surface of osteoclasts but whose identity and function
remain unknown. These data demonstrate the utility of
monoclonal antibody techniques in the identification of antigens that are preferentially expressed on osteoclasts, but
further studies are needed to determine the functional rate of
these antigens and their utility as specific markers for osteoclasts.
C. Calcitonin receptor
Perhaps the best differentiation marker for the osteoclast
is the calcitonin receptor. Calcitonin receptors are expressed
on a variety of cell types, including kidney and brain cells,
but do not appear to be expressed on macrophage polykaryons. The calcitonin receptor from several species have recently been cloned (37, 38), and at least two isoforms of the
calcitonin receptor have been identified. Ikegame and coworkers (38) have shown that one isoform, termed CIA, is
predominantly expressed in osteoclasts, and Goldring and
associates (37) have examined the expression of calcitonin
receptor isoforms in giant cells from human osteoclastomas.
Takahashi et al. (39) demonstrated that expression of the
calcitonin receptor occurs late during osteoclast precursor
differentiation, at a stage when precursor cells are committed
to the osteoclast lineage and are postmitotic. These studies
were performed with human bone marrow cells as a source
for putative osteoclast precursors. These workers have also
shown that calcitonin can down-regulate expression of the
calcitonin receptor mRNA. Down-regulation of calcitonin
receptor mRNA may in part explain the calcitonin escape
phenomenon, seen when osteoclasts are treated for long periods with calcitonin and are no longer responsive to calcitonin. Wada et al. (40) and Lee et al. (41) have confirmed that
calcitonin can down-regulate calcitonin receptor mRNA expression and that the calcitonin receptor is expressed on
CFU-GM
CD34 1
CD33*
Early
Precursor
Kn22*
CD11b"
My11*
Immature OCL
Mature OCL
CD4SA
Kn22*
CD11b*
VNR*
CTR 4
FIG. 2. Proposed surface phenotype of human osteoclasts and their
precursors. In this model, CFU-GM are the earliest identifiable osteoclast precursors. As the cells differentiate, CD34 expression is lost,
and the cells express the CD11B and CD45RA antigens, as well as the
Kn22 antigen, which is expressed on both early osteoclasts and monocytic precursors. The cells then differentiate to committed precursors
and express the vitronectin and calcitonin receptors. The immature
osteoclast still expresses antigens that are also present on precursors
and that are not expressed on mature osteoclasts, including CD11B
and CD45RA. Once the osteoclast is fully differentiated, it loses expression of CD45RA antigen.
311
osteoclasts and not on macrophage polykaryons or monocytes (42). Calcitonin receptors have been detected in preparations of isolated newborn rat osteoclasts in both multinucleated osteoclasts and subpopulations of mononuclear cells
present in bone marrow, but not in osteoblast-like cells (38).
Taylor et al. (43) have shown that between day 15 and 17 of
development, osteoclast progenitors in mouse marrow differentiate progressively from proliferative osteoclast progenitors lacking TRAP activity to cells that express TRAP and
calcitonin receptors. Similarly, Kurihara and co-workers (44)
have also shown that committed precursors for osteoclastlike cells formed in human marrow cultures expressed calcitonin receptors. Rodent osteoclasts express high numbers
of calcitonin receptors (45), expressing as many as 1,000,000
high affinity calcitonin receptors per cell. However, not all
bone-resorbing osteoclasts express calcitonin receptors.
Avian osteoclasts do not respond to calcitonin and do not
express calcitonin receptors. Thus, calcitonin receptor expression is not an absolute feature of resorbing osteoclasts.
However, calcitonin receptor expression appears to be one of
the best markers for distinguishing mammalian osteoclasts
from macrophage polykaryons.
D. PTH receptor
Until recently, it was thought that osteoclasts did not express PTH receptors. However, Agarwala and Gay (46) and
Teti et al. (47) have reported that murine or avian osteoclasts
express PTH receptors. The significance of PTH receptor
expression by mature osteoclasts is still unknown, since
highly purified mature osteoclasts do not respond to PTH
(48, 49). Hakeda et al. (50) have previously reported, using
immunocytochemical techniques, that osteoclast precursors
derived from murine hematopoietic blast cells expressed
parathyroid receptors and suggested that PTH may act directly on osteoclast precursors to induce their differentiation.
Similarly, Kurihara et al. (51) have reported that PTH is a
mitogen for highly purified human osteoclast precursors,
suggesting a direct effect of PTH on osteoclast precursors. In
contrast, coculture studies of osteoblasts with hematopoietic
cells that form osteoclasts (52), as well as studies on isolated
osteoclasts (7), have suggested that PTH appears to act indirectly through cells in the osteoblast lineage to stimulate
osteoclast formation and osteoclastic bone resorption. Thus,
it appears that osteoclast precursors may respond directly to
PTH, but mature osteoclasts, even though they may express
PTH receptors, do not respond directly to PTH.
E. pp60c-src
Osteoclasts have also been shown to express high levels of
pp60c-src, a non-receptor tyrosine kinase. In osteoclasts,
pp60c-src protein and kinase activity are expressed at levels
almost as high as in brain and platelets (53). This is in contrast
to other bone cells, which express very low levels of the
pp60c-src protein. Furthermore, when bone marrow cells are
induced to express an osteoclast phenotype by 1,25-dihydroxyvitamin D3, high levels of pp60c-src protein are also
expressed in these cells. Osteoclasts also express three other
src-like kinases, c-Fym, c-Yes, and c-Lyn (53), but these pro-
ROODMAN
312
teins do not play a role in osteoclastic bone resorption, since
blocking pp60c-src activity in the osteoclast inhibits bone
resorption. Using the techniques of homologous recombination, Soriano and co-workers (54) reported that transgenic
mice lacking the pp60c-src gene developed severe osteopetrosis. Furthermore, Tanaka and co-workers (55) showed that
osteoclasts expressed high levels of pp60c-src preferentially
on their ruffled border. Thus, although pp60c-src is expressed in a variety of cell types, expression of high levels of
pp60c-src is characteristic of the osteoclast, and src tyrosine
kinase activity is an important component of the bone resorption process.
IV. Origin of the Osteoclast
A. Hematopoietic origin of the osteoclast
Several lines of evidence have clearly shown that the osteoclast is hematopoietic in origin. The early studies of
Walker (56, 57) using parabiotic union of osteopetrotic mice
with normal littermates demonstrated that osteopetrosis
could be cured in these animals and that a marrow cavity
formed. Similarly, when one of a pair of parabiotic rats was
lethally irradiated, and the cells in a nonirradiated rat were
labeled with thorotrast (58), osteoclasts formed that were
derived from the nonirradiated animal. Studies using quailchick chimeras demonstrated that osteoclasts were host-derived (59), and transplantation studies in lethally irradiated
rodents and patients with osteopetrosis have confirmed that
the osteoclast is present in the hematopoietic tissues, such as
spleen, marrow, and peripheral blood (60-63). Taken together, these studies clearly demonstrated that osteoclast
precursors are present in the marrow and that circulating
osteoclast precursors are detectable in peripheral blood.
However, the hematopoietic lineage of the osteoclast still
remains a subject of controversy, although as noted below,
the majority of evidence favors CFU-GM, the granulocytemacrophage progenitor, as the earliest identifiable osteoclast
precursor.
B. Osteoclast precursors
The overwhelming majority of studies demonstrate that
the osteoclast precursor is a cell in the monocyte-macrophage
lineage. Young (64) was the first to describe putative osteoclast precursors. These cells were mononuclear, had ruffled
borders, and contained TRAP. However, this study did not
determine whether these cells were actually mononuclear
osteoclasts or osteoclast precursors. Using an in vivo model
of osteoclast formation in rats, Baron and co-workers (65)
demonstrated by histological techniques that mononuclear
cells that have a low nuclear-cytoplasmic ratio and an abundance of ribosomes invade sites of bone resorption. These
mononuclear cells initially contained nonspecific esterase, an
enzyme present in monocytes and not in mature osteoclasts;
as these mononuclear cells attach to the bone surface, they
differentiate into cells containing TRAP and eventually lose
nonspecific esterase activity. These TRAP-positive mononuclear cells eventually formed multinucleated osteoclasts in
Vol. 17, No. 4
these animals. These data demonstrated that the osteoclast
precursor was a cell in the monocyte-macrophage lineage.
Other studies performed in vivo and in vitro have further
supported that the osteoclast is derived from cells in the
monocyte-macrophage lineage. Fischman and Hay (66)
showed that in regenerating newt limbs, osteoclasts are
formed by fusion of cells that had a monocytic appearance
histologically, Similarly, Jee and Nolan (67) showed that
macrophages labeled with charcoal particles formed osteoclasts that also contained charcoal particles. Tinkler et al.
(68) injected [3H]-thymidine-labeled peripheral blood monocytes into syngeneic hosts treated with 1,25-dihydroxyvitamin D3 and showed that these labeled nuclei were present in
resulting osteoclasts. Zambonin-Zallone and co-workers (69)
have also reported that peripheral blood monocytes could
fuse with purified chicken osteoclasts, and Burger et al. (70)
were the first group to demonstrate the formation of osteoclasts from marrow cells in vitro. In their studies, murine
fetal bone rudiments that lacked osteoclasts were cocultured
in plasma clots with marrow cells and a source of macrophage colony-stimulating factor (M-CSF). Using histological
techniques, these workers demonstrated that osteoclasts
formed in these organ cultures and identified the precursor
for these osteoclasts as a cell in the monocytic lineage.
Oursler and co-workers (28) have shown that a monoclonal
antibody that identifies macrophages and macrophage
polykaryons also cross-reacts with osteoclasts, further supporting the monocyte-macrophage lineage as the osteoclast
lineage. More recently, Athanasou and co-workers (26) reported that macrophage antigens are also expressed on osteoclasts, and Kurihara et al. (44) have shown that osteoclastlike cells formed in human marrow cultures from highly
purified populations of CFU-GM, the granulocyte-macrophage progenitor, derived cells. In this culture system,
CD34+ marrow mononuclear cells are cultured in the presence of granulocyte-macrophage CSF (GM-CSF) for 7-10
days, and then the cultures are overlayered with 1,25-dihydroxyvitamin D3 for an additional 2 weeks. At the end of the
second culture period, four types of colonies are found in the
cultures: 1) granulocytic, 2) macrophage, 3) colonies of mixed
hematopoietic lineages, and 4) cells that have unique polygonal morphology. One hundred percent of these polygonal
cells react strongly with the 23c6 monoclonal antibody. The
cells express calcitonin receptors and fuse to form only osteoclast-like multinucleated cells; multinucleated cells
formed from these polygonal cells form resorption lacunae
on calcified matrices. Using these types of systems, we have
tested cytokines for their effects on both immature and committed mononuclear osteoclast precursors. All of the
multinucleated cells formed from these committed precursors contract in response to calcitonin, express the osteoclastic vacuolar proton pump, and react strongly with the 23c6
monoclonal antibody. Thus, these cells, which are derived
from CFU-GM, appear to be committed precursors for the
osteoclast. Schneider and Relfson (71) have also shown that
murine marrow enriched for CFU-GM give rise to osteoclasts. These data strongly support the hypothesis that osteoclast precursors are derived from cells in the monocytemacrophage lineage, with the CFU-GM as the earliest
identifiable precursor.
August, 1996
THE OSTEOCLAST
In addition to data suggesting immature monocytic cells
are osteoclast precursors, Udagawa and co-workers (72) reported that murine pulmonary alveolar macrophages and
peripheral blood monocytes could form osteoclasts when
cocultured with osteoblasts or marrow stromal cell lines.
These data suggest that, in addition to CFU-GM in bone
marrow, mature monocyte-macrophages can also form osteoclasts.
However, Hattersley and co-workers (73) and Lee et al. (74)
have suggested that a cell more primitive than CFU-GM is
the precursor for the osteoclasts and that the osteoclast lineage branches before these cells differentiate to CFU-GM.
Hattersley et al. (73), with the use of CFU-blast cultures,
showed that CFU-GM-derived cells formed only a small
percentage of the osteoclasts in these cultures and that more
primitive cells formed large numbers of osteoclasts. However, these workers did not identify a colony of pure osteoclast precursors using these techniques. Lee and co-workers
(74) also proposed a model in which the osteoclast is not
derived from CFU-GM. In this model, a recently described
osteoclast-stimulatory factor, CSF-O (osteoclast CSF) stimulates formation of colonies of TRAP-positive mononuclear
cells in semisolid marrow cultures. When GM-CSF was substituted as a source of colony-stimulating activity in these
cultures, no TRAP colonies formed. Only after prolonged
culture with GM-CSF could TRAP mononuclear cell colonies
be demonstrated in these cultures. Again, these data do not
negate the possibility that cells in the monocyte-macrophage
lineage may still be the precursor for the osteoclast. They
have not tested the possibility of treating CFU-GM-derived
cells with CSF-O to determine whether TRAP-positive colonies will form. Scheven et al. (75) have reported that murine
bone marrow cells enriched for hematopoietic stem cells can
form osteoclasts and that cultures of fetal bone rudiments
with pluripotent hematopoietic cell lines derived from
mouse bone marrow cultures such as FDCP1 cells form osteoclasts in vitro. However, these data do not exclude the
possibility that early osteoclast precursors differentiate to
CFU-GM before becoming osteoclasts. Thus, the majority of
studies support CFU-GM as the earliest recognizable precursor for the osteoclast. A schema for osteoclast differentiation that encompasses these different models is shown in
Fig. 3.
C. Characteristics of osteoclast precursors
Recently, the morphological and phenotypic characteristics of osteoclast precursors have been described. Takahashi
(unpublished observation) has shown in preliminary studies
that the earliest marker for cells in the osteoclast lineage
appears to be expression of MMP-9, which precedes expression of TRAP in CFU-GM-derived cells treated with 1,25dihydroxyvitamin D3. Lorenzo and co-workers (41) and
Findlay and associates (40), using murine marrow cultures,
reported that TRAP-positive mononuclear cells initially form
in these cultures and then acquire calcitonin receptor expression after treatment with 1,25-dihydroxyvitamin D3 for
several days. Calcitonin can down-regulate the calcitonin
receptor mRNA in these precursors (38). Kukita et al. (34)
have purified the precursors for osteoclast-like multinucle-
313
Models of Osteoclast Differentiation
Stem Cell
CFU-GM
OCL
CFU-O
OCL
Stem Cell
CFU-GM
Stem Cell
CFU-GM
Monocyte
OCL
FIG. 3. Models of osteoclast differentiation. Several models have
been proposed for osteoclast differentiation. In one model the hematopoietic stem cell gives rise to the granulocyte-macrophage progenitor cell, which in turn can give rise to osteoclasts. Alternatively,
CFU-GM give rise to monocytes and mature monocyte-macrophages
that can also form osteoclasts. In a third model, the hematopoietic
stem cell gives rise to a unique stem cell, CFU-O, the osteoclast
colony-forming cell that is distinct from cells in the granulocytemacrophage lineage. This cell then differentiates and fuses to form
mature osteoclasts.
ated cells formed in human marrow cultures. They demonstrated that the earliest osteoclast precursor is present in the
CD34-positive population of human marrow cells, and as it
differentiates, it acquires an antigen that is identified by the
monoclonal antibody Kn22. This early precursor also crossreacts with monoclonal antibodies MY9 and CD45RA and
expresses other myeloid antigens. Figure 2 shows the proposed surface phenotype for human osteoclasts precursors
as they differentiate toward the osteoclast lineage.
Wesolowski et al. (76) have recently reported the isolation
and characterization of highly purified prefusion murine
osteoclast precursors. The cells were mononuclear and expressed calcitonin receptors and mRNAs for MMP-9, carbonic anhydrase II, and high levels of pp60c-src protein.
These cells were purified by first removing stromal cells with
collagenase and then releasing the precursors with echistatin, a protein from snake venom that contains an RGD sequence. The precursors could resorb bone only if they were
cocultured with 1,25-(OH)2D3 and an osteoblastic cell line.
These data suggest that the early mammalian osteoclast precursor expresses TRAP and MMP-9 and is derived from
CFU-GM. As it differentiates and becomes committed to the
osteoclast lineage, these precursors express high levels of
pp60c-src, carbonic anhydrase, and calcitonin receptor.
V. Model Systems for Studying Osteoclast Formation
and Bone Resorption
Progress in understanding the molecular events that occur
during osteoclast differentiation and osteoclastic bone re-
314
ROODMAN
sorption has been difficult because, as noted above, osteoclasts are few in number, are fragile when isolated from
bone, and are difficult to isolate because they are embedded
in a calcified matrix; furthermore, no osteoclast cell lines are
available. To circumvent these problems, a variety of model
systems have been developed that form osteoclast-like cells
in vitro. These model systems have been used to identify
osteoclast precursors and to characterize their surface phenotype and for identification and characterization of factors
affecting osteoclast activity and formation.
A. Isolation of mature osteoclasts from bone
Several techniques have been developed for isolating mature osteoclasts from long bones. One of the most commonly
used sources for osteoclasts has been the endosteal surface of
chick long bones. In one approach, Osdoby and co-workers
(77) prepared osteoclasts from embryonic chick tibia by removing the marrow and then releasing the osteoclasts using
calcium-magnesium free buffers. The cell suspension was
sieved through nylon mesh to trap the large multinucleated
osteoclasts, and the osteoclasts were enriched by discontinuous density centrifugation. Cell preparations of 50-75%
pure osteoclasts could be obtained by these techniques, and
the osteoclasts could be cultured for 10 days and retain an
osteoclast morphology. However, only a small percentage of
these cells were viable and resorbed bone. A similar approach has been used by Zambonin-Zallone and co-workers
(78) using hypocalcemic egg-laying chickens. With their
methods, large numbers of osteoclasts can be isolated,
although the viability of these cells is still in question.
Chambers et al. (79) and Tezuka et al. (80) have isolated
osteoclasts from rats or rabbits and used these osteoclasts to
examine the effects of factors directly on osteoclasts. Osteoclasts can be isolated in large numbers from juvenile rabbit
long bones and can be highly enriched. These cell preparations have been used to generate osteoclast cDNA expression
libraries (81). However, these systems suffer from two shortcomings: 1) The majority of these cells may not be viable, and
2) they may contain mixed cell populations, making it difficult
to assess whether factors act directly or indirectly on osteoclasts.
Chambers (42) used isolated osteoclasts that are deposited
on bone slices as an assay of osteoclastic bone resorption. In
this assay, the number of resorption pits and the area
resorbed are quantified. Boyde and co-workers (82) have
argued that determining the volume of the pits is more important than measuring the area resorbed. In either case,
factors are applied to the cells, and the number of resorption
lacunae and the area or volume of the calcified matrices
resorbed are determined microscopically. However, such
techniques can give only limited information on the effects
of these factors on osteoclast differentiation and on the regulation of osteoclastic bone resorption, since no new osteoclasts form, and the cell populations tested are not homogeneous. The majority of studies using isolated osteoclasts
have suggested that most osteotropic factors act indirectly on
osteoclasts via the osteoblast or stromal cell. Furthermore,
there is a large variability in the capacity of these cells to
resorb bone, so that large numbers of bone slices are required
to obtain reproducible results.
Vol. 17, No. 4
B. Giant cell tumors as a model of osteoclasts
Several groups have used human giant cell tumors of bone
as a model of human osteoclasts. The giant cells from these
tumors express an osteoclast phenotype (32). They are extremely large, highly multinucleated, contain high levels of
TRAP, a marker enzyme for osteoclasts, have calcitonin receptors, contract in response to calcitonin, and form resorption lacunae on calcified matrices. These cells most likely
represent very highly activated osteoclasts. Giant cells react
strongly with the 23c6 monoclonal antibody developed by
Horton and co-workers (83), which cross-reacts with the
osteoclast vitronectin receptor. Using immunomagnetic bead
techniques, Ohsaki et al. (35) purified the 23c6-positive cell
fraction from disaggregated giant cell tumors and then further purified the multinucleated cells from mononuclear cells
in this fraction on Percoll gradients to 90-95% purity. These
techniques have permitted studies of osteoclast phenotype,
preparation of human osteoclast cDNA libraries (84), and
testing factors for their effects on osteoclasts.
C. In vivo models of osteoclast formation
As noted previously, Baron and co-workers (65) have developed an in vivo model for studying osteoclast formation in
rodent alveolar bone. Osteoclasts in alveolar bone are responsive to changes in the plasma concentration of PTH or calcitonin
and have enzymatic properties similar to those of osteoclasts in
other parts of the skeleton. This system has been used to study
osteoclast formation from mononuclear precursors. Bone resorption is induced by extracting molars from adult rats. The
absence of apposing teeth induces bone remodeling along the
periosteum of the mandible. The animals are then killed, in
groups, every 24 h, and the mandibles are processed for light
microscopy, ultrastructural studies, and cytochemical studies.
Although these techniques have provided important insights
into the sequence of osteoclast differentiation, they are extremely tedious, and it is difficult to isolate cells from these
preparations for additional studies.
Recently, we have reported an in vivo model to examine the
mechanism of action of osteotropic factors on various stages
of osteoclast formation (85). In this model system, mice are
either injected with a factor or implanted with Chinese Hamster Ovary cells, which have been transfected with the cDNA
for the factor of interest and constitutively express the factor
at high levels, to examine their effects on three specific stages
of osteoclast formation: 1) CFU-GM, the earliest identifiable
osteoclast precursor, as assessed by colony assays; 2) more
committed osteoclast precursors, as assessed by osteoclastlike cell formation in long-term marrow cultures derived
from these animals; and 3) mature osteoclasts as determined
by histomorphometry of calvariae from these animals. This
in vivo model system should help to dissect the site of action
in the osteoclast lineage of various cytokines involved in
normal bone remodeling, as well as in states of increased
bone turnover, such as osteoporosis.
D. Bone organ culture systems
Bone organ culture systems have provided and continue
to be useful bioassays for studying factors controlling oste-
August, 1996
THE OSTEOCLAST
oclastic bone resorption and in some cases, osteoclast formation. These organ culture systems have been useful for
identifying new agents that stimulate, as well as inhibit, bone
resorption. Among the most frequently used systems are the
fetal rat or mouse calvarial assay or fetal rat long bone system, in which pregnant rats or mice are injected with calcium-45, and the radius and ulna or the calvaria are dissected
from the fetuses or neonatal rodents. These bones are then
placed on membranes suspended on wire mesh and floated
over a chemically defined media, and various osteotropic
factors are then added to the media. After an appropriate
time period, the percent of calcium-45 released from the bone
relative to the total amount of calcium-45 in the bone fragment is determined. Light microscopic and ultrastructural
studies, which allow some estimate of the number of osteoclasts present over time, can also be performed on these
bone fragments. However, since these organ cultures represent mixed cell populations, determination of whether a factor(s) acts directly on osteoclasts or indirectly through marrow stromal cells or other cells present in the bone organ
culture is problematic. Furthermore, since most of these organ culture systems use fetal or neonatal tissues, these organ
culture systems may not reflect the behavior of osteoclasts in
adult animals.
E. Bone marrow culture systems
Since, as noted above, the osteoclast is hematopoietic in
origin, bone marrow culture techniques have been applied to
studies of osteoclast development. In these cultures osteoclast-like multinucleated cells form. Burger and co-workers
(70) were the first to describe a marrow culture system that
formed osteoclasts. They used fetal bone rudiments that were
devoid of osteoclasts and cocultured them with marrow as
a source of osteoclast precursors in the presence of M-CSF.
The effects of a factor(s) in this system was determined by
histomorphometry, which is time consuming and labor intensive. Lowik and co-workers (86) used fetal mouse metacarpals and metatarsals for such studies. In their culture
system, fetal metacarpals only contain osteoclast precursors,
while the fetal metatarsals contain preformed osteoclasts.
When factors are added to either the metacarpal or metatarsal cultures, one can then distinguish whether the factor
stimulates osteoclast formation and /or also acts on mature
osteoclasts. This system also requires histological evaluation
of bone rudiments.
Testa and co-workers (87) were the first to modify the
classic Dexter long-term marrow culture systems to form
osteoclast-like cells. In their original work, multinucleated
cells formed in feline bone marrow cultured in a-MEM with
horse serum in the absence of any osteotropic factors. These
multinucleated cells were not well characterized but were
thought to be osteoclast-like because of their morphology.
Ibbotson and co-workers (88) further characterized this culture system, demonstrating that osteotropic factors could
modulate formation of these osteoclast-like cells appropriately and that the precursor for these cells was a cell in the
monocyte-macrophage lineage. Since those initial studies,
marrow culture systems that form osteoclast-like cells have
been extended to include culture systems using mouse mar-
315
row, in which TRAP-positive multinucleated cells that extensively resorb bone form in 5 to 6 days (89), baboon marrow
cultures (90), and human marrow cultures (91-93) in which
osteoclast-like cells form in 3 weeks. In a typical human
marrow culture, nonadherent marrow mononuclear cells are
cultured at 106 cells/ml in the presence of 1,25-dihydroxyvitamin D3 (10~8 M) for 3 weeks. Few, if any, multinucleated
cells formed in the first week of culture, and the majority of
multinucleated cells formed during the second week of culture. Maximum numbers of osteoclast-like multinucleated
cells are formed in these cultures after 3 weeks, and by 4
weeks the cells begin to detach from the plastic surface.
Multinucleated cells from these cultures fulfill the functional
criteria of osteoclasts. They are TRAP positive, react with the
23c6 monoclonal antibody, express calcitonin receptors, contract in response to calcitonin, and form resorption lacunae
on calcified matrices such as sperm whale dentine, although
the resorption lacunae are not as prominent or as frequent as
with murine marrow cultures. These human marrow culture
systems have been used to identify the mechanisms of action
of a variety of factors on osteoclast formation. During the first
week of the cultures, the cells are in a growth phase in which
the early precursors proliferate, and during the second and
third week of culture, these cells differentiate and fuse to
form multinucleated cells. Factors that stimulate proliferation of the precursors can be assessed by scoring the number
of [3H]thymidine-labeled nuclei incorporated into multinucleated cells in cultures treated for the first 24 h with [3H]thymidine. Factors that enhance fusion increase the number of
nuclei per multinucleated cell (Fig. 4).
Udagawa et al. (72) have used mouse spleen cells as a
source of osteoclast precursors. When these cells are cocultured with an appropriate bone marrow-derived stromal cell
(MC3T3-G2-PA6 or ST2) or with primary mouse calvarial
cells in the presence of dexamethasone and 1,25-dihydroxyvitamin D3, multinucleated cells formed. Osteoclastlike cells formed in these cultures contain TRAP and have
enhanced cAMP production in response to calcitonin. Calcitonin receptors were also demonstrated on the multinucleated cells formed in these cocultures by autoradiographic
techniques with [125I]calcitonin (94). Numerous resorption
lacunae were formed when spleen cells and ST2 cells were
cocultured on sperm whale dentine in the presence of 1,25(OH)2D3 and dexamethasone. Cell to cell contact with stromal cells or osteoblasts was absolutely required for osteoclast
formation in this culture system. This culture system has
been adapted to form large numbers of osteoclast-like cells
that can be released by collagenase when the cultures are
done on collagen gel-coated plates (94, 95).
Alvarez and colleagues (96) have used avian mononuclear
phagocytes from bone marrow to form osteoclast-like cells in
vitro. These investigators used uniform populations of nonspecific esterase-positive adherent marrow mononuclear
cells isolated from hypocalcemic hens. The adherent mononuclear cells had been isolated by adherence to plastic and
then cultured in a-MEM containing 5% FCS and chicken
serum with 5 /xg/ml cytosine-D-arabinofuranoside. Multinucleated cells that were generated over the 7 days of these
cultures had ruffled borders, formed resorption lacunae on
devitalized bone, and showed a cAMP response to prosta-
Vol. 17, No. 4
ROODMAN
316
Proposed Sites of Action of Factors on Osteoclast Formation and Activity
CFU-GM
Early
Precursor
Committed
Precursor
Immature OCL
Dying OCL
Mature OCL
Estrogens
Bisphosphonates
M-CSF
Proliferation
Fusion
•U-
Activation
Apoptosis
FIG. 4. Stages of osteoclast differentiation. During the osteoclast differentiation process, early precursors for the osteoclasts proliferate and
become postmitotic. At this stage, they undergo fusion and form multinucleated osteoclasts. Factors can act at either or both stages of osteoclast
differentiation. For example, IL-6 appears to stimulate osteoclast precursor proliferation, while factors such as PTH can stimulate both
proliferation and differentiation of osteoclast precursors. Factors that stimulate osteoclast activity are shown above the arrows, while factors
that inhibit osteoclast activity are shown below the arrows.
glandin E2. This marrow culture system allows collection of
large numbers of mononuclear precursor cells that express
an osteoclast phenotype and is useful for studies examining
osteoclast formation and bone resorption. However, it is
unclear how cytokines affect the formation of these cells,
although this system can provide large numbers of highly
purified osteoclasts and their precursors.
F. Osteoclast-like cell lines
Since an osteoclast precursor cell line is currently not available, investigators have used hematopoietic cell lines to form
osteoclast-like cell lines. Hattersley et ah (97) have reported
the generation of osteoclast-like cells from murine hematopoietic multipotential cell lines in vitro. Using FDCPA4 cells
cocultured with bone marrow stromal cells in the presence
of 1,25-dihydroxyvitamin D3, they demonstrated that these
pluripotent hematopoietic cells can form low numbers of
multinucleated cells that express an osteoclast phenotype
and form resorption lacunae on calcified matrices. Similarly,
Hagenaars and co-workers (98) used FDCP-C2-GM cells,
another multipotent hematopoietic murine cell line, and
demonstrated that when these cells are cocultured with fetal
bone rudiments, some osteoclast-like cells formed. Yoneda et
ah (99) have also used HL-60 cells, a human promyelocytic
cell line, to form osteoclast-like multinucleated cells. They
cultured HL-60 cells in methylcellulose containing conditioned media from a squamous cell carcinoma tumor cell line
that induced hypercalcemia in vivo. The cultures were then
treated with 1,25-(OH)2D3 to form osteoclast-like multinucleated cells. However, the number of osteoclast-like cells
formed from HL-60 cells was extremely low. Brandi and
co-workers (100) have also used a human myelo-monocytic
leukemia cell line as a model for osteoclast precursors but,
although the cells have some characteristics of osteoclasts,
they do not resorb bone (100). Chambers et ah (101) recently
reported that cells from transgenic mice, which contain a
transgene composed of the SV40 large T antigen driven by
an MHC promoter, can be used to produce osteoclasts in
vitro. They demonstrated that a hematopoietic cell line could
be derived from these mice that formed osteoclast-like cells.
Only a very small percentage of the cells formed osteoclasts,
similar to their results with the FDCP cell line. Several marrow stromal cell lines that supported osteoclast differentiation from spleen cells or marrow cells were also derived from
these mice. Thus, cells derived from these transgenic mice do
not appear to be committed to the osteoclast lineage but,
rather, to be earlier precursors that have the potential to form
small numbers of osteoclasts in vitro. None of these cell lines
described above form large numbers of osteoclasts that are
suitable for biochemical or molecular biological studies.
Recently, Shin and co-workers (102) reported that the murine macrophage cell line BDM-1 could form osteoclast-like
cells when cocultured with primary osteoblasts and 1,25(OH)2D3 for 14 days. Subclones of this cell line that did not
express the F4/80 macrophage antigen were the only cells
that could form osteoclast-like cells, and only 5-9% of the
cells could form osteoclasts. As noted above, development of
an osteoclast cell line and an osteoclast precursor cell line
would be a major advance in studies of the cell biology of the
osteoclast.
In an attempt to develop an osteoclast cell line, we have
cloned the promoter for TRAP, a marker enzyme for the
osteoclast, from a murine genomic library (9). We have fused
this promoter to the cDNA for SV40 large T antigen, and
made transgenic mice, to target the T antigen to the osteoclast
(11). These transgenic mice develop osteopetrosis and have
large numbers of transformed osteoclasts. The osteoclasts
contain mitotic figures and transformed nuclei, and many of
them are aneuploid. The mice develop osteopetrosis, but the
osteoclasts can respond appropriately to IL-1 and PTH-related peptide (PTHrP), with increased bone resorption. Thus,
T antigen can be targeted to the mature osteoclast by these
techniques, and it may be possible to isolate osteoclast or
osteoclast precursor cell lines from these TRAP-Tag mice.
However, osteoclasts formed in these mice undergo apoptosis, suggesting that a combination of oncogenes may be required to immortalize the osteoclast or its precursor.
Thus, a variety of model systems with which to examine
osteoclast formation and bone resorption have been developed. Marrow culture systems or spleen-stromal cell systems
appear to be the best models for examining osteoclast formation, while bone organ culture systems or isolated oste-
August, 1996
THE OSTEOCLAST
oclasts are better for testing the effects of factors on mature
osteoclasts. The isolated osteoclast pit assay has yielded useful information, but the lack of purity of the cell populations
tested and variability of the assay are problematic.
VI. Factors That Affect Osteoclast Function and
Formation
A. Systemic hormones
1. PTH. PTH can induce bone resorption, increase bone formation, and inhibit osteoblast activity depending on its mode
of administration. When it is administered continuously,
bone formation is suppressed, and osteoclastic bone resorption is increased. The major mechanism that has been proposed for the action of PTH on osteoclasts has been an indirect one. In this model, PTH acts on the osteoblast, which
in turn produces a mediator such as M-CSF or interleukin-6
(IL-6) that stimulates osteoclastic bone resorption and formation (52). Hyperparathyroid patients have increased osteoclastic bone resorption, which results in bone loss accompanied by marrow fibrosis. PTH causes a marked increase in
bone resorption in bone organ culture systems (103) and
stimulates osteoclast formation in both murine and human
marrow culture systems (89, 91). In addition, Lorenzo et al.
(104) have reported that PTH acts predominantly on a postmitotic cell in the osteoclast lineage. Using our recently described in vivo model of osteoclast formation, we demonstrated that PTH and PTHrP appear to act on the more
differentiated osteoclast precursor, rather than the early proliferative precursor (105). This more differentiated precursor
then fuses to form osteoclasts. Cytokines such as IL-1, transforming growth factor-a (TGFa), tumor necrosis factor-a,
and IL-6 can enhance the effects of PTH on osteoclast formation and osteoclastic bone resorption. IL-6, for example,
can stimulate proliferation of early osteoclast precursors, and
PTH then induces the differentiation and fusion of these
precursors to form multinucleated osteoclasts (106). Furthermore, PTH and PTHrP enhance calcium reabsorption by the
kidney. In patients with tumors that secrete PTHrP, this
enhanced renal calcium reabsorption, in addition to the increased osteoclastic bone destruction, contributes to the development of hypercalcemia. Thus, although it is unclear
whether PTH acts directly on osteoclasts, it has major effects
on osteoclast formation and osteoclastic bone resorption in
vivo and in vitro and can act in concert with other osteotropic
factors to enhance osteoclast activity.
317
reabsorption, as well as enhance osteoclastic bone resorption
stimulated by PTH. Mice treated with 1,25-(OH)2D3 develop
hypercalcemia, and analogs of 1,25-(OH)2D3 have variable
effects on osteoclast activity (109).
3. Prostaglandins. The effects of prostaglandins on osteoclast
formation and osteoclastic bone resorption may be dependent on the dose administered and the assay system used.
Prostaglandins are stimulators of osteoclastic bone resorption in bone organ culture systems and stimulate osteoclast
formation in murine marrow cultures (89). However, PGE2
inhibits osteoclastic bone resorption and formation in human
systems (7,110). Chambers et al. (7) have reported that isolated osteoclasts treated with PGE2 contract and pull away
from the bone surface in a similar fashion as osteoclasts
treated with calcitonin, thereby inhibiting bone resorption.
Tashjian and associates (111) have reported that a variety of
factors that stimulate osteoclastic bone resorption in the
mouse calvarial organ culture system do so by generating
prostaglandins. The data suggest PGE2 may be an important
second message for cytokines that enhance bone resorption.
Recently, Gallwitz et al. (112) showed that other arachidonic acid metabolites such as the peptidoleukotrienes, as
well as 5-hydroxyeicosatetraenoic acid, stimulate isolated
osteoclasts to resorb bone. These 5-lipoxygenase metabolites
are produced by stromal cells isolated from giant cell tumors
of bone, suggesting that they may serve as a vehicle for
stromal cell enhancement of osteoclast activity. These arachidonic metabolites may also play an important role in bone
resorption in areas of chronic inflammation, in addition to
the effects of tumor necrosis factor-a (TNFa) and IL-1 (see
below).
4. Calcitonin. Calcitonin is a peptide hormone secreted by the
parafollicular cells of the thyroid gland and is a potent inhibitor of osteoclastic bone resorption. It acts at multiple
stages in the osteoclast lineage, including inhibition of osteoclast formation, and inhibits the bone-resorbing capacity
of mature osteoclasts. Calcitonin receptors are expressed on
committed osteoclast precursors and appear to be a differentiation marker for the mature osteoclast (39). Calcitonin
down-regulates expression of calcitonin receptors in osteoclast precursors and mature osteoclasts by inhibiting expression of the mRNA for its receptor (39,41). Calcitonin acts
on osteoclasts by stimulating adenylcyclase activity and
cAMP accumulation, which results in immobilization of the
osteoclasts and contraction of the osteoclasts away from the
bone surface. Osteoclasts continually exposed to calcitonin
2. Calcitriol. Metabolites of vitamin D3 are potent stimulators can escape from the effects of calcitonin. The mechanism
responsible for this escape phenomenon is unclear but may
of osteoclastic bone resorption and osteoclast formation. The
be due to the effects of calcitonin on expression of the calmost active metabolite, 1,25-dihydroxyvitamin D3, acts as a
citonin receptor and on receptor synthesis at the transcripfusigen for committed osteoclast precursors (44). It is untional level. Calcitonin has been used effectively as a therlikely that 1,25-dihydroxyvitamin D3 acts on mature osteapeutic modality in patients with Paget's disease and
oclasts directly, since mature osteoclasts do not express vitamin D receptors (107). Feyen et al. (108) have reported that patients with the hypercalcemia of malignancy. In contrast to
patients with hypercalcemia of malignancy, patients with
1,25-dihydroxyvitamin D3 can induce IL-1 production and
Paget's disease can respond to calcitonin for prolonged peIL-6 production by osteoblasts, factors that stimulate osteriods of time (113). However, the molecular mechanisms
oclastic bone resorption. Furthermore, 1,25-dihydroxyvitaresponsible for the prolonged responsivity of pagetic ostemin D3 increases calcium absorption from the gut and can act
oclasts to calcitonin have yet to be defined.
in conjunction with PTH to stimulate renal tubular calcium
318
ROODMAN
B. Factors that enhance osteoclast activity
1. IL-1. IL-1 is a cytokine produced by monocyte-macrophages and marrow stromal cells and probably osteoclasts
that can stimulate bone resorption in vitro and in vivo. IL-1
induces bone resorption and osteoclast-like cell formation in
murine and human marrow cultures (114, 115). Boyce and
co-workers (116) demonstrated that local injection of IL-1
over the calvaria of mice increased osteoclastic bone resorption within 24 h, and this was followed by increased bone
formation over the next 3-4 weeks. The bone formation, but
not the bone resorption, could be prevented by treating the
mice with indomethacin. These data demonstrate that IL-l's
effects on osteoclasts are not mediated by prostaglandins.
Similarly, Sabatini et al. (117) reported that infusion of IL-1
in normal mice induced hypercalcemia and bone resorption.
Recently, Uy et al. (85) have used an in vivo model of
osteoclast formation to examine the systemic effects of IL-1
on the different stages of osteoclast development. IL-1 induced hypercalcemia and enhanced the growth and differentiation of CFU-GM, the earliest identifiable osteoclast precursor. It also increased the number of more committed
mononuclear osteoclast precursors and stimulated mature
osteoclasts to resorb bone. The data demonstrate that IL-1
affects all stages of osteoclast development and may explain
its potent effects on bone turnover in vivo.
IL-1 also appears to mediate in part the effects of marrow
stromal cells on osteoclast formation. Takahashi et al. (118)
established a human bone marrow stromal cell line (Saka
cells) by infecting normal marrow adherent cells isolated
from semisolid marrow cultures with a recombinant adeno/
SV40 virus. Coculture of Saka cells with human marrow
mononuclear cells enhanced formation of osteoclast-like
multinucleated cells in human marrow cultures. These osteoclasts expressed calcitonin receptors and formed resorption lacunae on dentine slices. PCR analysis of the Saka cells
detected expression of mRNAs for IL-1/3 and IL-6. Addition
of neutralizing antibodies to IL-1/3 or IL-6 blocked the stimulatory effects of Saka cells on osteoclast-like cell formation.
IL-1 has also been implicated in several pathological conditions associated with increased bone loss. It is produced by
several tumors associated with hypercalcemia, such as squamous cell carcinoma and lymphoma (119,120). Freshly isolated marrow cells derived from some patients with myeloma produced IL-1/3, and the bone-resorbing activity
present in culture media from these marrow cell isolates
could be neutralized by IL-1/3 antibodies (121, 122).
2. CSFs. CSFs are hematopoietic growth factors that induce
the clonal growth of hematopoietic progenitors in vitro and
in vivo. They are produced by macrophages, stromal cells,
endothelial cells, and T lymphocytes in the marrow microenvironment. Since the osteoclast is hematopoietic in origin,
it is not surprising that these factors may act as stimulatory
factors for osteoclast formation.
M-CSF, also called CSF1, appears to play an important role
in osteoclast development. Studies in the op/op osteopetrotic
mouse, which has a point mutation in the M-CSF gene (123)
and has severe osteopetrosis due to an absence of osteoclasts,
have clearly shown an important role for M-CSF in murine
osteoclast development. Injection of M-CSF into op/op mice
Vol. 17, No. 4
improves the skeletal sclerosis in these animals although
some authors have suggested (124) that some residual osteosclerosis remains. Furthermore, Nilsson and co-workers
(125) have reported that the osteopetrosis in op I op mice improves with age, suggesting that M-CSF is required for early
osteoclast development, but that other cytokines can mimic
the effects of M-CSF at later times.
M-CSF appears both to stimulate the proliferation and
differentiation of osteoclast precursors (126). When mouse
osteoblasts are cocultured with mouse spleen cells as a source
of osteoclast precursors, osteoclast-like multinucleated cells
form in response to 1,25-dihydroxyvitamin D3. Addition of
anti-M-CSF or anti-M-CSF receptor antibody to these cultures, either during the first 4 days or the last 2 days of
culture, inhibited osteoclast-like cell formation (126). These
data suggest that M-CSF is required for both proliferation
and differentiation of osteoclast precursors to mature osteoclasts. Others have suggested the major effect of M-CSF is
to stimulate proliferation of murine osteoclast precursors
(127). In vitro experiments have shown that M-CSF can induce osteoclast formation in murine marrow cultures if the
cultures are treated sequentially with M-CSF followed by
1,25-dihydroxyvitamin D3 (128). M-CSF is also produced by
murine stromal cell lines and mouse calvarial cells and is the
factor responsible for their effects on osteoclast formation in
coculture systems. M-CSF can also stimulate osteoclast-like
cell formation in human marrow cultures if they are treated
sequentially for the first week with M-CSF followed by 1,25dihydroxyvitamin D3 (129).
In addition to its direct effects on osteoclast precursors,
M-CSF may also affect mature osteoclasts. The c-fms receptor,
which is the M-CSF receptor, has been demonstrated on
mature osteoclasts (130). Weir and co-workers (130) have
shown that PTH and PTHrP induce M-CSF production by
osteoblasts, as well as cells from the osteoblast cell line
ROS17/2.8. These data suggest that M-CSF may be a mediator of the osteoblast-osteoclast interactions induced by PTH
and PTHrP. Fuller and co-workers (131) have also identified
a role for M-CSF in maintaining the survival and chemotactic
behavior of mature osteoclasts. In their studies, M-CSF prevented apoptosis of osteoclasts, enhanced osteoclast motility,
and inhibited bone resorption.
The effects of other CSFs are not as clear as those for
M-CSF. GM-CSF stimulates the growth of osteoclast precursors and induces osteoclast formation when human bone
marrow cultures are treated sequentially with GM-CSF followed by 1,25-dihydroxy vitamin D3 (129). However, GMCSF by itself has no effect on osteoclastic bone resorption,
and it can inhibit osteoclast formation and osteoclastic bone
resorption in murine marrow cultures (132,133). Takahashi
and associates (128) have examined the effects of CSFs on
murine osteoclast formation in vitro in a coculture system;
their studies showed that all these factors can stimulate the
growth of osteoclast precursors when the cultures are treated
sequentially with CSFs followed by 1,25-(OH)2D3 but, when
added simultaneously with 1,25-(OH)2D3, inhibit the effects
of 1,25-(OH)2D3 on osteoclast formation. These data suggest
that the simultaneous addition of CSFs with 1,25-(OH)2D3
may result in the early bipotent precursor for osteoclasts and
macrophages differentiating to mature monocytes rather
August, 1996
THE OSTEOCLAST
than osteoclasts, while sequential addition of CSFs followed
by l/25-(OH)2D3 may allow the CSFs to expand the precursor
pool and then permit l/25-(OH)2D3 to act unopposed and
induce osteoclast differentiation.
319
calcium homeostasis and osteoclastic bone resorption in vivo
(106), and soluble IL-6 receptor must be added to murine
marrow cultures to induce osteoclast formation in vitro (144).
IL-6 receptors have been demonstrated on human osteoclasts
(35), and IL-6 can stimulate osteoclast-like cell formation in
3. TGFa. TGFa, a polypeptide that is partially homologous human marrow cultures in the absence of added IL-6 recepto epidermal growth factor, is produced by several solid
tors (143).
tumors associated with the hypercalcemia of malignancy.
IL-6 may also act as an autocrine/paracrine factor proTGFa can stimulate osteoclastic bone resorption in murine
duced by osteoclasts in Paget's disease of bone (145). Osteorgan cultures (131) by binding the epidermal growth factor
oclast-like multinucleated cells formed in human marrow
receptor (134). TGFa is a proliferative factor that stimulates
cultures from patients with Paget's disease actively express
the growth of early osteoclast precursors but by itself has no
IL-6 transcripts and release IL-6 into their conditioned media;
CSF-like activity (135).
this media stimulated osteoclast-like cell formation in normal
TGFa induces osteoclastic bone resorption and hypercalmarrow cultures. Furthermore, patients with Paget's disease,
cemia in nude mice and increases osteoclast-like cell formabut not normal subjects, have elevated levels of IL-6 in their
tion in human marrow cultures (134, 135). Immunohistomarrow plasma and their peripheral blood.
chemical studies by Hiraga et al. (136) demonstrated the
We recently examined the effects of antisense constructs to
presence of TGFa in bone adjacent to areas of osteoclast
IL-6 on the bone-resorbing capacity of highly purified giant
activation and bone resorption induced by a metastatic hucells from giant cell tumors of bone to help define the role of
man melanoma cell line in nude mice. Furthermore, TGFa
IL-6 in human osteoclastic bone resorption (146). IL-6 levels
can act synergistically with PTHrP to stimulate bone resorpin conditioned media from highly purified giant cells were
tion in vivo (137). Thus, TGFa can stimulate osteoclast forelevated. Treatment of these giant cells with IL-6 antisense
mation and bone resorption both in vitro and in vivo and may constructs or neutralizing antibodies to IL-6 (35) caused a
act in concert with PTHrP also produced by tumors to cause
4-fold decrease in IL-6 levels and significantly decreased the
hypercalcemia in patients with cancer.
number of resorptive lacunae formed and the area of the
dentine resorbed. These observations demonstrated that IL-6
4. Tumor necrosis factors. Both TNFa and TNF/3 (lymphotoxin) may play an important role in the bone-resorptive process of
markedly stimulate the formation of osteoclast-like multinuhuman osteoclasts.
cleated cell in human marrow cultures (115). TNF can also
Interestingly, IL-6 has been found to induce a marked loss
affect the activity of mature osteoclasts. Thomson et al. (138,
of calcitonin binding sites on normal T lymphocytes at con139) have shown that bone resorption is stimulated by incentrations known to be active on bone metabolism (147).
cubating mature osteoclasts cocultured with osteoblastic
IL-6 may also play an important role in other states of incells with IL-1 or TNF.
TNF potentiates the effects of IL-1 on osteoclast formation creased bone destruction, such as multiple myeloma bone
disease and Gorham-Stout disease, also known as "disap(115). Garrett et al. (140) demonstrated that myeloma cell
pearing bone syndrome," where it has recently been implilines released a bone-resorbing activity into their conditioned
cated (Fig. 5) (148).
media, which was TNF/3 (lymphotoxin), and that neutralizing antibodies to lymphotoxin blocked the bone resorption
6. Interleukin-11 (IL-11). IL-11 is a newly described cytokine
in bone organ cultures induced by media conditioned by
that shares many of the biological properties of IL-6. It stimthese myeloma cells. However, increased levels of TNF/3
ulates hematopoiesis, induces immunoglobulin production
have not been consistently found in patients with myeloma
or in an in vivo model of human myeloma bone disease (141). and acute phase protein synthesis, and inhibits adipogenesis
(149). In addition, Girasole and co-workers (150) have imTumor necrosis factors appear to stimulate both proliferation
plicated
IL-11 as a cytokine critical for osteoclast developand differentiation of osteoclast precursors and may be inment.
IL-11
is produced by a variety of cell types, including
volved in the pathogenesis of hypercalcemia of malignancy.
stromal
cells
and osteoblasts, and the effects of IL-11 as well
Yoneda and co-workers (142) have shown that the hyperas IL-6 are mediated by gpl30 signal transduction compocalcemia, induced by a squamous cell carcinoma cell line
nents. IL-11 can stimulate the formation of osteoclasts when
implanted into nude mice, is in part mediated by TNFa. In
murine
bone marrow cells are cocultured with calvarial cells.
this model system, TNFa was produced by host macroOsteoclasts
formed in the presence of IL-11 show a high
phages in response to the tumor. These data suggest that host
degree
of
ploidy
and form resorption lacunae on calcified
factors may further aggravate the hypercalcemia induced by
matrices.
Furthermore,
addition of a neutralizing antibody to
tumor-derived factors.
IL-11 suppressed osteoclast development induced by 1,255. IL-6. IL-6 is a 26,000-dalton cytokine produced by many dihydroxyvitamin D3, PTH, IL-1, or tumor necrosis factor in
murine marrow cultures, suggesting that IL-11 may be a
cells in the bone microenvironment including marrow strocommon mediator of the effects of these cytokines on ostemal cells, monocyte-macrophages, osteoclasts, and osteooclast formation. The effects of IL-11 on osteoclast developblasts. It induces osteoclast formation from osteoclast prement appear to be mediated by inducing prostaglandin syncursors (86, 143). However, its role in osteoclast activity is
thesis since indomethacin blocked the effects of IL-11 on
controversial. It does not appear, by itself, to be a potent
osteoclast development. Consistent with these observations
osteotropic factor in murine systems in vivo (106). IL-6 poare the findings of Romas and co-workers (151), who showed
tentiates the effect of other hormones such as PTHrP on
ROODMAN
320
VoZ. 17, iVo.
Paget's
Disease
GorhamStout
Disease
I
L
-
6
Myeloma
Osteoporosis
FIG. 5. IL-6, a potential mediator of osteolytic bone diseases. IL-6 has been implicated in a number of osteolytic processes, including Paget's
disease of bone, multiple myeloma, postmenopausal osteoporosis, and Gorham-Stout disease ("disappearing bone disease"). In Paget's disease,
IL-6 may act as an autocrine/paracrine factor that stimulates osteoclast formation and osteoclastic bone resorption. IL-6 is also a growth factor
for myeloma cells and may enhance bone resorption. In animals, after ovariectomy, stromal cells have been reported to produce IL-6, which
in turn enhances osteoclast formation and activity. In Gorham-Stout disease IL-6 is present in the serum, and it may have a role in increased
bone destruction in these patients.
that 1,25-(OH)2D3 induced IL-11 production in osteoclast
cocultures and that 1,25-dihydroxyvitamin D 3 and PTH can
induce IL-11 production by osteoblasts. These data suggest
that a variety of osteotropic factors can induce osteoblastic
production of IL-11, and Romas and co-workers (151) have
also shown that osteoclasts express abundant IL-11 receptor
mRNA. However, at present, it is unknown whether IL-11 is
a physiological mediator of bone resorption or only increases
osteoclast formation during inflammatory conditions.
C. Inhibitory
factors
1. Transforming growth factor-ft (TGF/3). TGF/3 has been p r o -
posed as one of the key factors involved in coupling bone
formation to previous bone resorption (153). It is secreted by
osteoblasts and osteoclasts and may act as an autocrine factor
stimulating osteoblastic bone formation through enhanced
chemotaxis, proliferation, and differentiation of committed
osteoblasts (153).
TGFjS is secreted as a dimer composed of 12.5-kDa subunits noncovalently associated with one or more polypep7. Oxygen free radicals. Garrett and co-workers (152) reported tides to form a latent complex of higher molecular mass.
that oxygen free radicals can stimulate osteoclast formation
Latent TGF/3 can be experimentally activated by proteinase
and osteoclastic bone resorption. These investigators demtreatment, acidification, or denaturation to dissociate the maonstrated that when oxygen free radicals were generated
ture factor from the latent complex. Bone contains very high
adjacent to the bone surfaces in vitro or in vivo, osteoclasts
levels of latent TGF/3 and is the largest source of TGF/3 in the
formed. Furthermore, they showed that PTH- and IL-1-stimbody.
ulated bone resorption was inhibited by superoxide disPfeilschifter and Mundy (154) have reported that ostemutase, an enzyme that depletes tissues of superoxide anoclasts can activate latent TGF/3. Oursler (155) also showed
ions. Nitroblue tetrazoleum (NBT) staining of resorbing bone
that osteoclasts expressed TGF/3 mRNA and that latent TGF/3
was used to identify the cell types producing free radicals in
can be activated by osteoclasts. He concluded that osteoclasts
resorbing bone. These studies showed that the osteoclasts
may secrete proteinases that activate latent TGFjS into the
were the only cells that stained with NBT, and that calcitonin
extracellular space and that TGF/3 may be an autocrine factor
for osteoclasts as well.
decreased NBT staining in osteoclasts. These results suggested that cytokine stimulation of osteoclasts induces oxyTGF/3 is a potent inhibitor of osteoclastic bone resorption.
gen derived free radical production, and that oxygen free
It modulates both osteoclastic bone resorption and migration
radicals may be intermediaries in the formation and activaand osteoclast differentiation in bone organ cultures (156).
Chenu et ah (157) have also shown that TGF/3 inhibits both
tion of osteoclasts by cytokines.
August, 1996
THE OSTEOCLAST
the proliferation and fusion of human osteoclast precursors.
Ikeda et al. (158) have suggested that TGF/3 may regulate
normal osteoclast activity since mRNA levels of TGF/3 are
decreased in rat bone after ovariectomy. These data have
suggested that TGF0 may be a "coupling factor" that is
released and activated during osteoclastic bone resorption
and then inhibits osteoclast formation and activity and induces new bone formation (154).
2. y-Interferon. 7-Interferon is a potent inhibitor of bone resorption in vitro (159) and suppresses the formation and
maturation of osteoclasts (160). Tohkin et al. (161) examined
the effects of 7-interferon on the humoral hypercalcemia of
malignancy in nude mice bearing lower jaw tumors, in which
PTHrP was responsible for inducing hypercalcemia. Mice
were injected with 7-interferon for 5 days before the establishment of hypercalcemia, and 7-interferon treatment delayed the increase in plasma calcium concentrations. 7-Interferon treatment also abolished the formation of
multinucleated osteoclast-like cells from bone marrow cells
of these mice ex vivo. The data suggest that 7-interferon
suppresses the formation of osteoclasts in vivo, resulting in
decreased plasma calcium concentrations.
Gowen and Mundy (159) have also shown that 7-interferon blocks the bone-resorbing effects of IL-1 and tumor
necrosis factor in bone organ cultures. 7-Interferon appears
to be a more effective inhibitor of bone resorption stimulated
by IL-1 and tumor necrosis factor than bone resorption stimulated by PTH or 1,25-dihydroxyvitamin D3. The basis for
this differential effect of 7-interferon on IL-1- and PTH-stimulated bone resorption is unknown. Possibly, IL-1 and TNFa
effects are mediated by different second message components than PTH and 1,25-(OH)2D3, and 7-interferon only
affects the former. Kurihara and Roodman (162) have also
shown that a-interferon can also inhibit osteoclast formation
in human marrow cultures, suggesting that the interferons as
a class are inhibitors of bone resorption.
321
nitric oxide produces a rapid contraction and detachment of
osteoclasts from cell surfaces and inhibits bone resorption by
osteoclasts. Furthermore, inhibition of nitric oxide synthase
in rats in vivo is accompanied by increased bone resorption.
Nitric oxide synthase activity has been detected in normal rat
osteoclasts by both Northern blot and immunocytochemical
studies (168). Lowik and co-workers (169) reported that osteoblast-like cells in fetal mouse long bone explants can also
produce nitric oxide. 7-Interferon, together with TNFa and
lipopolysaccharide, induced nitric oxide synthesis by osteoblast-like UMR106 cells and inhibited bone resorption in fetal
mouse bone organ cultures. TGF/3 inhibited the stimulatory
effects of these cytokines on nitric oxide production. These
data suggest that nitric oxide production by osteoblasts may
represent an important regulatory mechanism of osteoclast
activity, especially in inflammatory conditions associated
with cytokine release.
5. Sex steroids. Estrogen is one of the major inhibitors of
osteoclast formation. With ovariectomy, increased osteoclastic bone resorption and osteoclast formation occur. Osteoblasts contain estrogen receptors, and Oursler and co-workers (31, 170) demonstrated recently that osteoclasts also
contain estrogen receptors. The mechanism responsible for
the increased bone turnover with estrogen deficiency is still
a point of contention, but IL-6, IL-1, and TNFa have been
implicated in this process.
IL-6 production by bone marrow stromal cells is suppressed by 17/3-estradiol in vitro. Jilka and co-workers (171)
reported that estrogen loss after ovariectomy in mice increased the number of CFU-GM, enhanced osteoclast development in ex vivo cultures of marrow, and increased the
number of osteoclasts in trabecular bone. These changes
were prevented by administration of a neutralizing antibody
to IL-6. These findings suggest that IL-6 may be involved in
the increased bone resorption in postmenopausal osteoporosis, with estrogen loss resulting in an IL-6-mediated stim3. IL-4. IL-4 is a product of activated T cells that affects both ulation of osteoclasts. Similar effects of androgen depletion
immunological and hematopoietic processes. Shioi et al. (163) on IL-6 production have also been reported by these investigators (172). In recent studies with animals that do not have
reported that IL-4 inhibited the formation of osteoclasts from
a
functional IL-6 gene, Poli et al. (173) and Lowik and assomurine bone marrow cells cocultured with stromal cells, and
ciates
(174) have shown that these animals have increased
Watanabe et al. (164) and Riancho et al. (165) showed that IL-4
bone
turnover
but, when ovariectomized, do not show furinihibited bone resorption in organ cultures.
ther
enhanced
bone
turnover compared with nonovariectoNakano et al. (166) examined the in vivo effects of IL-4 on
mized
controls.
Similarly,
it has been reported that mice
spontaneous and PTHrP-stimulated mouse osteoclast foroverexpressing
the
gene
for
the TNF-soluble receptor do not
mation. EC-GI cells, which produce PTHrP, were implanted
have increased bone turnover after ovariectomy (175). Thus,
into nude mice, and after the mice became hypercalcemic,
the cytokine(s) responsible for increased bone turnover and
they were treated with IL-4. Continuous infusion of IL-4
postmenopausal osteoporosis is still unclear and remains an
returned the calcium levels to normal. Histomorphometric
area of active investigation. However, as noted above, IL-1
analysis revealed that IL-4 inhibited osteoclast formation in
and/or TNFa may be potential mediators for the bone loss
these mice as demonstrated by a decrease in osteoclastic
seen with ovariectomy (176).
surfaces and in the number of osteoclasts per normal bone
Kimble and co-workers (177) have shown that adminissurface. However, transgenic mice overexpressing IL-4 detration of IL-1 receptor antagonist to animals that have unvelop an osteopenic syndrome that is similar to osteoporosis
dergone ovariectomy decreases bone loss and bone resorp(167). These data suggest that high levels of IL-4 may also
tion. Estrogen deficiency was necessary to unveil the boneinhibit bone formation, as well as bone resorption.
sparing effects of the IL-1 receptor antagonist because
4. Nitric oxide. Nitric oxide is a potent multifunctional signal administration of the IL-1 receptor antagonist to control animals for 4 weeks before ovariectomy did not induce any
molecule that has widespread actions in a variety of tissues
change in their bone mineral density. Kitazawa et al. (178)
including bone. Brandi and co-workers (168) showed that
322
Vol. 17, No. 4
ROODMAN
have recently reported ovariectomy-increased bone marrow
stromal cell secretion of IL-1 and the formation of TRAP(+)
osteoclast-like cells in bone marrow cultures treated with
1,25-(OH)2D3 in vitro. The effects of ovariectomy on osteoclast
formation in vivo and in vitro were blocked by treatment of
the mice with an anti-IL-1 receptor antagonist and antiTNFa. Both IL-1 and TNFa had to be blocked to prevent bone
loss after ovariectomy, suggesting that TNF works in concert
with IL-1 to induce bone loss in this model system. Thus, it
is unclear whether IL-6, IL-1, TNFa, or all three cytokines are
responsible for the increased osteoclast formation and bone
resorption in animals after oophorectomy. Most likely, these
cytokines represent part of a cytokine cascade whereby
TNFa may induce IL-6 or IL-1.
6. Bisphosphonates. Bisphosphonates are derivatives of pyrophosphates that inhibit formation, delay aggregation, and
slow the dissolution of calcium phosphate crystals and bind
calcium phosphates strongly. This is the basis for the use of
these compounds as inhibitors of osteoclastic bone resorption and as tracer compounds for nuclear medicine studies
of bone. Bisphosphonates block bone resorption in both organ cultures and in vivo. Different bisphosphonates have
different potencies, e.g. etidronate is 100- to 1000-fold less
potent than alindronate in its ability to inhibit bone resorption. The mechanism of the inhibitory action of bisphosphonates on bone resorption is not completely understood.
When bone slices or dentine slices are treated with bisphosphonates before the addition of osteoclasts, osteoclastic bone
resorption is inhibited. A possible mechanism that has been
proposed in this action is that the osteoclast is inhibited
directly by taking up bisphosphonates from bone (179).
Fleisch and co-workers (180), however, have suggested that
bisphosphonates inhibit osteoclastic bone resorption by stimulating secretion of an inhibitor of osteoclast recruitment or
survival by osteoblasts. Bisphosphonates have been used to
treat hypercalcemia of malignancy, Paget's disease, and osteoporosis and are being actively used in the treatment of
increased bone destruction in patients with myeloma (181).
Thus, there are a variety of factors that stimulate or inhibit
osteoclast formation and bone resorption. Systemic hormones can have stimulatory or inhibitory effects, and locally
acting factors such as the cytokines can have potent effects
on osteoclast formation and bone resorption. Table 2 summarizes the activity of several of these factors that regulate
osteoclast activity.
VII. Fusion Factors Involved in Osteoclast Formation
Factors that mediate the fusion of mononuclear osteoclast
precursors to form multinucleated osteoclasts are just beginning to be identified. PTH/PTHrP and 1,25-(OH)2D3 can
induce fusion and differentiation of osteoclast precursors.
Recently, Mbalaviele and co-workers (182) reported that Ecadherin, a member of the family of homophilic calciumdependent cell adhesion molecules, is involved in the fusion
of osteoclast precursors. Immunocytochemical studies of human and mouse bone, using monoclonal antibodies to Ecadherin, demonstrated expression of E-cadherin in osteoclasts. Neutralizing monoclonal antibodies to E-cadherin
TABLE 2. Factors that regulate osteoclast activity
Factor
Formation
Resorption
PTH
(+)
(+)
Calcitriol
(+)
(+)
PGE2
(+) (murine)
(+) (murine)
PGE2
(-) (human)
(—) (human)
IL-1
(+)
(+)
M-CSF
(+)
(-)
TGFa
(+)
(+)
TNFa and TNF/3
(+)
(+)
(+)
IL-6
(+)
?
IL-11
(+)
Calcitonin
(-)
(-)
TGF/3
(-)
(-)
-y-Interferon
(-)
(-)
IL-4
(-)
(-)
Sex steroids
(-)
(-)
Bisphosphonates
?
(-)
Stimulation of osteoclast formation and/or resorption is denoted by
(+); inhibition of formation and/or resorption is denoted by (-).
decreased the number of TRAP-positive multinucleated cells
formed in murine marrow cultures but did not inhibit the
proliferation of these cells or their attachment to the culture
dish. Furthermore, synthetic peptides containing the cell adhesion recognition sequence of cadherins also decreased
TRAP-positive multinucleated cell formation in murine marrow cultures. These results suggest that E-cadherin may be
involved in the fusion of osteoclast precursors.
VIII. Osteoclast Adhesion Molecules
Attachment of osteoclasts to the bone surface is required
for bone resorption and is mediated by integrins, including
the vitronectin receptor. Osteoclasts also express the al, )31,
and a v j31 integrins (183). Integrin molecules are a family of
membrane glycoproteins formed by heterodimers of a- and
/3-subunits. The vitronectin receptor (a v )33) binds to the
extracellular matrix at a tripeptide arginine-glycine-aspartic
acid (RGD) recognition site. The a v )33 integrin colocalizes
with vinculin and tailin in the podosomes of osteoclasts at the
attachment site to bone. Podosomes are thought to represent
dot-like contact adhesion sites for osteoclasts (184) and have
been suggested to play a central role in osteoclast attachment
to bone. The vitronectin receptor appears to mediate the tight
attachment of osteoclast to bone matrix, especially to osteopontin, a bone matrix protein containing the RGD sequence. Osteotropic factors such as 1,25-dihydroxyvitamin
D3 and IL-4 can induce expression of the a v j83 integrin gene.
1,25-(OH)2D3 can up-regulate both the a v and j33 genes (185,
186), while IL-4 increases /33 mRNA levels by transactivating
the j33 gene (187). Antibodies against the osteoclast vitronectin receptor can block the bone-resorbing capacity of osteoclasts (188). However, the vitronectin receptor is not located at the site of bone resorption but is in close proximity
to it, suggesting that the capacity of the 23c6 antibody to
block bone resorption results from its effects on osteoclastic
attachment to bone rather than direct effects on bone resorption (188).
Recently, van der Pluijm and co-workers (189) demonstrated in fetal mouse bone organ cultures that integrins are
also involved in osteoclast formation. Synthetic RGD pep-
August, 1996
THE OSTEOCLAST
tides that block integrin binding inhibited fusion of mononuclear precursors to form multinucleated osteoclasts. These
data suggest that adhesion molecules may be important in
the fusion process for osteoclast precursors to form mature
osteoclasts, as well as in osteoclastic bone resorption.
IX. The Role of the Bone Microenvironment in
Osteoclast Formation
Suda and co-workers (72,190) reported that, in a coculture
* system using spleen cells or marrow cells as a source of
osteoclast precursors, marrow stromal cells or osteoblasts
were absolutely required for osteoclast formation. However,
stromal cell lines developed from mouse bone marrow display heterogeneity in their capacity to support osteoclasto* genesis (191). Stromal cells or osteoblasts must be able to
produce M-CSF to induce osteoclast formation, since marrow
stromal cells or osteoblasts from op/op mice that lack M-CSF
do not support osteoclast differentiation (192). Soluble MCSF could not replace the requirement for stromal cells in this
* coculture system, suggesting that membrane-bound M-CSF
. or other factors produced by marrow stromal cells or osteoblasts are absolutely required for murine osteoclast development from precursors. However, it is unclear whether
marrow stromal cells or osteoblasts are also absolutely required for human osteoclast formation. Kurihara and coworkers (44) reported that highly purified early osteoclast
precursors derived from CFU-GM can be treated with 1,25(OH)2D3 in the absence of marrow stromal cells or osteoblasts
and form osteoclast-like cells that express calcitonin receptors and form resorption lacunae on calcified matrices. Similarly, Pacifici and co-workers (193) recently reported that
CD34-positive cells isolated from peripheral blood cells and
cultured in the presence of GM-CSF and IL-1 formed
multinucleated cells that could form resorption lacunae on
calcified matrices in the absence of stromal cells. These data
• suggest that, at least in human systems, stromal cells or
osteoblasts may not be absolutely required for osteoclast
formation. However, human marrow stromal cells can enhance osteoclast formation. Takahashi et ah (118) developed
a human marrow stromal cell line, Saka cells, that can induce
osteoclast-like cell formation in human marrow cultures.
This cell line produces both IL-1 and IL-6, and addition of
neutralizing antibodies to IL-1 or IL-6 to the cocultures blocks
the capacity of the Saka cells to induce osteoclast formation.
These data demonstrate there are species differences in the
requirements for cells from the bone microenvironment to
induce osteoclast formation. Microenvironmental elements
may be absolutely required for murine osteoclast formation
* but only serve to enhance human osteoclast formation.
X. The Osteoclast as a Secretory Cell
Recent evidence has suggested that the osteoclast is a
secretory cell that produces factors which can stimulate its
own formation and activity. As noted above, early studies in
our laboratory demonstrated that the osteoclast-like cells
formed in marrow cultures from patients with Paget's disease produce IL-6, and that IL-6 can stimulate osteoclast
323
formation and bone resorption. Reddy et ah (146) have used
antisense constructs to IL-6 and demonstrated that these
antisense constructs decreased IL-6 production and the boneresorbing capacity of giant cells. This was the first demonstration that antisense constructs to a factor produced by
osteoclasts could inhibit the bone-resorbing capacity of osteoclasts. Recently, Pike and co-workers (194) reported that
murine osteoclasts also produce IL-6 and express IL-6 receptors and the components of the IL-6 signaling pathway.
In addition to producing IL-6, osteoclast-like giant cells
from giant cell tumors of bone also produce IL-1. Oursler
(155) also has shown that osteoclasts synthesize and secrete
latent TGFjB. This investigator used highly purified avian
osteoclasts to examine TGF/3 synthesis and demonstrated,
both by Northern blot analysis and metabolic labeling studies, that TGF/3 was produced. The principal form of TGF/3
produced by the osteoclasts was TGF/32. Furthermore, nearly
all the TGF/3 that was secreted by the osteoclasts was activated. When presented with exogenous latent TGF/3, these
avian osteoclasts could activate TGF/3 from a variety of
sources, consistent with the previous observations of
Pfeilschifter and Mundy (154), who showed that latent TGF0
could be activated during osteoclastic bone resorption. Since
TGF/3 is a negative regulator of osteoclastic bone resorption
and osteoclast formation, these data suggest that osteoclasts
produced both stimulatory and inhibitory factors that can
regulate their own formation and activity.
Demonstration that osteoclasts produce autocrine factors
represents an important addition to our understanding of the
regulation of normal osteoclast formation and activity. Takahashi et ah (195) prepared a mammalian cDNA expression
library generated from highly purified human osteoclast-like
multinucleated cells formed in bone marrow cultures and
screened this library for autocrine factors that enhance osteoclast-like cell formation. The cloning strategy for identifying autocrine factors produced by osteoclasts is shown in
Fig. 6.
In the initial screening, Annexin II was identified, and
purified recombinant Annexin II significantly increased osteoclast-like cell formation in human bone marrow cultures
in the absence of 1,25-dihydroxyvitamin D3 and enhanced
the bone-resorptive capacity of 1,25-dihydroxy vitamin D3 in
bone organ cultures. Annexin II was thought originally to be
exclusively an intracellular protein that was an inhibitor of
phospholipase A2. We have shown that Annexin II is secreted by osteoclasts from giant cell tumors and is present in
RNA from pagetic bone. Annexin II can stimulate osteoclast
formation in vitro in both human and murine systems. Annexin II appears to be a proliferative factor that stimulates the
growth of early osteoclast precursors (196), rather than inducing fusion and differentiation of the cells. Nesbitt and
Horton (197) have reported that Annexin II is also expressed
on the surface of osteoclasts, and inhibition of Annexin II
with a specific antibody to Annexin II blocked bone resorption by isolated osteoclasts.
Expression cloning techniques have been used to identify
a variety of other factors produced by osteoclasts. We have
recently identified a novel osteoclast stimulatory factor,
which we named OSF-1, that can stimulate formation of
human and murine osteoclast-like cells in vitro (198). This
ROODMAN
324
Vol. 17, No. 4
pc DNA 1
Vector
Cloning
poly (A)+ RNA
Cell Culture
Expression
Vector
Cloning
23c6+ cell
counting
•
Conditioned
Media -
OCL Precursors
TRAP
Assay
Transient cDNA
Expression in 293 cells
Long-term Human
Bone Marrow Culture
FIG. 6. Expression-cloning strategies for identifying autocrine factors produced by osteoclasts. Human marrow mononuclear cells were
cultured in the presence of 1,25-dihydroxyvitamin D3 for 3 weeks until
they formed osteoclast-like multinucleated cells that express the vitronectin receptor. These multinucleated cells were then purified by
immunomagnetic bead techniques using the 23c6 monoclonal antibody that identifies the vitronectin receptor, and poly(A) RNA was
isolated from these highly purified osteoclast-like cells. The poly(A)
RNA was converted to cDNA and cloned into the Agtll vector, and one
round of amplification was done. These cDNAs were then cloned into
the pcDNAl expression vector, and the clones were then transiently
transfected into 293 cells. Conditioned media from 293 cells were
tested for their capacity to stimulate osteoclast-like cell formation in
both human and murine marrow culture systems. Osteoclast-like cell
formation was determined by measuring TRAP activity in the lysates
and confirmed by counting the number of multinucleated cells.
factor has a novel peptide sequence that is not related to any
known cytokine and is currently being characterized. cDNA
libraries also have been prepared from rabbit osteoclasts (81)
and highly purified giant cells from giant cell tumors of bone.
Baron and co-workers (84) have used these techniques to
clone a component of vacuolar proton pump that plays an
important role in hydrogen export to the extracellular space
beneath the ruffled border in osteoclasts (199). Thus, it appears that autocrine regulation of osteoclast formation and
activity is an important area for future investigation (Fig. 7).
OCL
FIG. 7. Autocrine regulation of osteoclasts. A number of autocrine
factors have been identified that are produced by the osteoclasts and
can enhance both osteoclast formation from osteoclast precursors, and
osteoclastic bone resorption by mature osteoclasts. These include
IL-1, IL-6, and a newly described osteoclast-stimulatory factor-1
(OSF-1). In addition, negative autocrine regulatory factors are produced by the osteoclasts. The latent form of TGF/3 is produced by
osteoclasts and is then activated. TGF/3 can inhibit both osteoclastic
bone resorption and osteoclast formation.
*
crease in TRAP activity and osteoclastic bone resorption activity in the osteoclasts that formed as compared with controls. These data suggest that prolonged expression of c-fos
can enhance osteoclast differentiation. Hoyland and Sharpe '
(202) have also shown that c-fos protooncogene expression is
up-regulated in pagetic osteoclasts. Using immunocytochemical techniques with c-fos antibodies, these workers
showed increased protein expression in pagetic osteoclasts
and demonstrated increased expression of c-fos mRNA in
osteoclasts from Paget's patients by in situ hybridization.
Taken together, these data suggest that c-fos plays a critical
role in differentiation of osteoclast precursors at the branch y
point where the osteoclast lineage diverges from the macrophage lineage.
B. pp60c-src
pp60c-src Is a nonreceptor tyrosine kinase that is expressed in most cells. The osteoclast expresses very high
levels of c-src. Targeted disruption of the c-src gene in mice
results in osteopetrosis (54). These mice have osteoclasts, but
XI. Protooncogenes Involved in Osteoclast
the osteoclasts lack ruffled borders and do not form resorpDifferentiation and Bone Resorption
tion lacunae (203). In resorbing osteoclasts, pp60c-src is localized to the ruffled border. Furthermore, transplantation of
A. c-fos
fetal liver cells into irradiated src-minus recipients demon- -*
c-fos, A gene normally associated with osteosarcomas, ap- strated that the inherent defect is with the osteoclast and not
with the bone marrow microenvironment (204). Further suppears to be a key regulator of osteoclast differentiation.
port for the importance of pp60c-src activity in osteoclastic
Grigoriadis and co-workers (200) have shown, using the
bone resorption is the work of Yoneda et al. (205), who
techniques of homologous recombination, that mice lacking
the c-fos protooncogene develop osteopetrosis. The fos mu- showed that herbimycin, a pp60c-src tyrosine kinase inhibitor, blocked osteoclastic bone resorption in vitro and hypertant mice have a block in differentiation at the branch point
calcemia induced by IL-1 in vivo. In murine marrow cultures,
between monocyte-macrophages and osteoclasts. Bone marPTH increased the number of osteoclasts, pp60c-src tyrosine
row transplantation rescues the mice from osteopetrosis, and
ectopic c-fos expression can overcome this block in osteoclast kinase activity, and src protein, while short exposure to caldifferentiation. Mice lacking the c-fos protooncogene have citonin decreased pp60c-src protein and pp60c-src protein
tyrosine kinase activity, as well as the number of osteoclasts
normal macrophage differentiation but lack osteoclasts. Con(206). These data suggested that a src tyrosine activity in
sistent with this observation are the results of Miyauchi and
osteoclasts can be regulated by osteotropic hormones. Thus, i
co-workers (201) who reported that osteoclast precursors, as
pp60c-src plays an important role in regulating osteoclast
well as mature multinucleated avian osteoclasts, show constitutive expression of c-fos mRNA. Transfection of c-fos activity. Studies of the disruption of pp60c-src activity suggests that pp60c-src may have a specific function in ostecDNA into avian osteoclast precursors induced a 2-fold in-
August, 1996
THE OSTEOCLAST
oclasts. Home and co-workers (53) have shown that three
other src-like kinases, c-fyn, c-yes, and c-lyn, are also ex. pressed in osteoclasts but cannot compensate for absence of
pp60c-src in src-deficient mice (53).
XII. Mechanisms of Osteoclastic Bone Resorption
Osteoclasts resorb bone by secreting proteases that dis. solve the matrix and acid that releases bone mineral into the
extracellular space under the ruffled border. Osteoclast secretion of hydrogen ion can be modulated by regulators of
osteoclastic bone resorption. For example, PTH and prostaglandin E2 increase acid secretion by osteoclasts (207), while
calcitonin decreases acid secretion. Microelectrode-based pH
measurements at the ruffled border have shown pH levels as
* low as 3-4 (208). Acid secretion by the osteoclast requires a
proton pump. At least three different types of proton pumps
have been implicated in the acidification process, but the
strongest evidence suggests that a vacuolar type proton
pump, which is similar to the kidney H+-ATPase, is involved
* in osteoclastic bone resorption (199). This proton pump ap, pears to transport protons against a concentration gradient.
Protons are supplied for the proton pump by the action of
several enzymes including carbonic anhydrase II (209). The
critical importance of carbonic anhydrase II in the osteoclast
has been shown by studies in patients with a congenital
absence of this enzyme and osteopetrosis (210). Similarly, the
carbonic anhydrase inhibitor acetozolamide (211) can inhibit
osteoclastic bone resorption. Lysosomal hydrolases, which
are active at acid pH, resorb the organic matrix. As noted
above, the osteoclast contains high levels of MMP-9, which
may act in concert with collagenase to degrade the collagen
matrix. The recent discovery of cathepsin O, a cysteine proteinase that can degrade collagen in addition to cathepsin B
and L, suggests that the osteoclast secretes enzymes that may
directly digest collagen.
XIII. Osteoclast Apoptosis
Although much is known about factors regulating osteoclast formation and osteoclast activity, little information is
available about factors involved in osteoclast senescence.
• Osteoclasts undergo apoptosis, programmed cell death that
is characterized by nuclear and cytoplasmic condensation
and fragmentation of nuclear DNA into nucleosomal-sized
units. This fragmentation of DNA can be demonstrated by
gel electrophoresis or by in situ labeling of the fragmented
DNA through incorporation of labeled deoxyoligonucleotides at the single-stranded ends of these DNAs, using terminal deoxynucleotidal transferase. Hughes and co-workers
(212) have recently developed an in vitro system for examining apoptosis in osteoclasts. In this system, murine marrow
is cultured in the presence of 1,25-dihydroxyvitamin D3 until
osteoclast-like cells form. The media is then changed, and the
factor of interest is added 24-48 h later. Osteoclasts released
• into the supernatant are counted and their morphology examined. Using this culture system, Hughes et al. have shown
that TGFj3 can induce apoptosis of mature osteoclasts. TGF/3
can also induce apoptosis in a variety of other tissues, such
325
as liver and hematopoietic cell lines, and will cause cell death
in tumor cell lines derived from prostate, liver, and kidney
tumors (213). More recently, Hughes and co-workers have
shown that sex steroids, including estrogen and testosterone,
can promote osteoclast apoptosis both in vitro and in vivo
(214). Addition of estradiol, tamoxifen, or testosterone to
murine marrow cultures increased osteoclast apoptosis 2- to
3-fold compared with controls. Similarly, sex steroids also
increased osteoclast apoptosis in vivo in ovariectomized or
orchiectomized mice treated with either estradiol or testosterone, respectively. Bisphosphonates, which block bone resorption, also can induce apoptosis in osteoclasts (215), while
osteoclast-stimulatory factors, such as 1,25-dihydroxyvitamin D3 and PTH, inhibit induction of osteoclast apoptosis in
vitro (216). These data suggest that regulation of osteoclast
life span plays an important role in the normal bone-remodeling process to either enhance or inhibit osteoclastic bone
resorption. Cytokines that enhance osteoclast activity do so
in part by increasing osteoclast life span, and factors that
inhibit osteoclast activity appear to induce osteoclast apoptosis in addition to blocking osteoclast formation and bone
resorption.
XIV. Abnormalities in Osteoclast Function
A. Paget's disease
In Paget's disease of bone, the primary cellular abnormality resides in the osteoclast. In Paget's disease there are large
numbers of abnormal osteoclasts that are increased in size
and have increased numbers of nuclei (up to 100) per
multinucleated cell (2). These osteoclasts have increased levels of TRAP and contain viral and cytoplasmic viral-like
inclusions (217). These nuclear inclusions cross-react with
antibodies directed against paramyxoviral nucleocapsid proteins (218), and in situ hybridization studies have shown that
mRNAs for measles virus or canine distemper virus are
present in these osteoclasts (219, 220). Furthermore, abnormalities in osteoclast precursors have also been detected in
in vitro studies with marrow from affected bones from patients with Paget's disease (221). Osteoclast precursors are
increased in number in marrow aspirates and are hyperresponsive to 1,25-dihydroxyvitamin D3, forming osteoclastlike cells in vitro at concentrations of 1,25-(OH)2D3 that are
one tenth that required for normal osteoclast-like cell formation in vitro. Furthermore, osteoclasts in Paget's patients
express high levels of IL-6 and IL-6 receptors, as well as the
c-fos protooncogene (203, 222). In addition, these osteoclast
precursors from Paget's patients appear to be hyperresponsive to the marrow microenvironment and show enhanced
growth when cocultured with normal or pagetic marrow
stromal cells (221).
The marrow microenvironment also appears to be abnormal in Paget's disease, since coculture of stromal cells from
Paget's patients with osteoclast precursors from normal subjects results in enhanced growth of normal osteoclast precursors (221). Stromal cell lines from Paget's patients also
appear to induce enhanced osteoclastic bone resorption in
vitro compared with normal stromal cell lines, further suggesting an abnormality in the bone microenvironment in
326
ROODMAN
Paget's patients (223). Recently, we have reported the presence of measles virus nucleocapsid transcripts in freshly
isolated marrow mononuclear cells from patients with
Paget's disease and demonstrated by sequence analysis that
there are mutations present in a highly localized region of the
C-terminal portion of this nucleotide sequence (224). In addition, circulating mononuclear cells in patients with Paget's
disease, as well as osteoclast precursors, also express measles
virus nucleocapsid transcripts (225). These data suggest that
Paget's disease is a slow viral disease, but the role of the virus
and the pathological process or its identity remain to be
determined.
B. Hypercalcemia of malignancy
Similar to patients with Paget's disease, patients with hypercalcemia of malignancy also have increased osteoclastic
bone resorption. However, in contrast to pagetic osteoclasts,
the osteoclasts in patients with hypercalcemia of malignancy
or multiple myeloma are normal in morphology (Table 3).
These osteoclasts are increased in number due to the actions
of cytokines or hormones produced by tumors or the host in
response to tumors that enhance osteoclast formation and
osteoclastic bone resorption. Tumors produce a variety of
factors that enhance osteoclast formation, including PTHrP,
IL-1, IL-6, as well as yet to be identified factors produced by
myeloma cells. The hypercalcemia in these patients and increased bone destruction is due to increased osteoclast activity. These patients respond to inhibitors of osteoclast activity, such as calcitonin or bisphosphonates, and tumor cells
themselves do not directly resorb bone (226).
C. Osteopetrosis
The absence of osteoclast function, either because there is
decreased osteoclast formation or because of impaired osteoclast activity, can result in osteopetrosis. As noted previously, there are several models of osteopetrosis in rodents,
including the op/op mouse in which osteoclast formation is
markedly inhibited because of a point mutation in the M-CSF
gene and the c-fos knockout mouse in which macrophages
but not osteoclasts are formed, as well as other models in
which there is decreased osteoclast formation (227). There are
also conditions in which osteoclast activity is impaired (228).
For example, in pp60c-src knockout mice, osteoclasts are
formed but they do not resorb bone. Similarly, patients with
congenital carbonic anhydrase II deficiency also forms osteoclasts, but again these osteoclasts are unable to produce
protons that are secreted into the attachment site under the
ruffled border, so that bone resorption does not occur. Key
and co-workers (229) have shown that y-interferon or calcitriol can enhance osteoclast activity in some patients with
osteopetrosis. Since the osteoclast is a hematopoietic cell,
bone marrow transplant studies have been done in children
with severe osteopetrosis, and some of these patients have
been cured by bone marrow transplant (63). Thus, any condition that impairs osteoclast formation or osteoclast activity
can result in osteopetrosis. Consistent with these observations are our studies in transgenic mice (11) in which large
T antigen has been directed to the osteoclast using the TRAP
Vol. 17, No. 4
promoter. These animals develop osteopetrosis because the
large T antigen interferes with the differentiated function of
the osteoclast.
D. Osteoporosis
Osteoporosis can result from increased osteoclast activity
or decreased formation of bone by osteoblasts. In postmenopausal osteoporosis, there appears to be increased osteoclast
activity and bone resorption, while in senile osteoporosis •
there appears to be a defect in osteoblast activity. Studies in
patients with postmenopausal osteoporosis have not provided a definitive answer as to the role of cytokines in this
process. As noted above, after ovariectomy in rodent models,
cytokines such as IL-6, IL-1, or TNFa have been implicated
in the increased osteoclast activity in these animals. Estrogens suppress osteoclast activity, and ovariectomy results in
stimulation of IL-6 and IL-1 and TNFa production in these
animals. Manolagas and co-workers (230) have shown that
estrogen regulates IL-6 promoter activity and may affect IL-6
production even though the IL-6 promoter does not contain
an estrogen-responsive element. Pfeilschifter and co-workers '
(231) have demonstrated increased IL-6 levels in patients <
with postmenopausal osteoporosis, and Pacifici et al. (232)
have shown that peripheral monocytes from postmenopausal patients with increased bone turnover produced more
IL-1 than age-matched controls. However, Riggs and associates (233) were unable to find any increase in IL-6 or IL-1
production in bone marrow plasma or serum from patients
with postmenopausal osteoporosis. At present, it is unclear
whether these cytokines are actively playing a role in patients ,
with postmenopausal osteoporosis, and until local levels of
cytokines can be accurately measured in these patients, it will
be difficult to ascertain their role in this process.
XV. Summary and Conclusions
Much has been learned about the cell biology and molecular biology of the osteoclast in the last 5 yr. The osteoclast
appears to be derived from CFU-GM, the committed monocyte-granulocyte precursor cell. This cell then differentiates
into more committed precursors for the osteoclast. The role
of the marrow microenvironment appears to be critical for
murine osteoclast formation, although in human systems it
appears to be nonessential but acts to enhance osteoclast
formation and resorption. The osteoclast has been shown to
be a secretory cell capable of producing both stimulators and
inhibitors of osteoclast formation and resorption. The identification of the role of protooncogenes, such as c-fos and
pp60c-src, in osteoclast differentiation and bone resorption
has provided important insights into the regulation of normal osteoclast activity. Studies such as these should help us
to dissect the pathophysiology of abnormal osteoclastic activity, such as seen in hypercalcemia of malignancy, osteopetrosis, and Paget's disease of bone. Future research is
needed to further delineate the signaling pathways involved
in osteoclastic bone resorption in response to cytokines and
hormones, as well as to identify the molecular events required for commitment of multipotent precursors to the osteoclast lineage. Development of osteoclast cell lines may be
August, 1996
THE OSTEOCLAST
possible and would greatly enhance our understanding of
the biology of the osteoclast. Utilization of current model
systems to examine the effects of cytokines and hormones on
osteoclast precursors in vitro and in vivo and the ability to
obtain large numbers of highly purified osteoclasts for production of osteoclast cDNA libraries should lead to important new discoveries in osteoclast biology.
20.
21.
22.
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