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Molecular mechanisms of bone resorption by the osteoclast.

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THE ANATOMICAL RECORD 224:317-324 (1989)
Molecular Mechanisms of Bone Resorption
by the Osteoclast
Yale University School of Medicine, Departments of Orthopedics, Cell Biology, and
Medicine, New Hauen, Connecticut 06510
The osteoclast is a multinucleated cell that is actively engaged in
the synthesis of lysosomal enzymes, their vectorial transport toward the apical
membrane, and the secretion of these enzymes a t its apical pole. These secreted
enzymes are targeted to the apical ruffled-border membrane by mechanisms that
involve cation-independent mannose-6-phosphate receptors. These receptors bind
to an enzyme-linked mannose-6-phosphate recognition marker in the Golgi complex, and the enzyme-ligand-receptor complex, carried within small coated transport vesicles, dissociates upon reaching the low pH established in the bone-resorbing compartment by the osteoclast. The apical bone-resorbing compartment is
sealed off by the attachment of the osteoclast to the calcified matrix and is actively
acidified by the osteoclast. The plasma membrane of the cell is divided into distinct
domains. The apical membrane at the ruff led-border shares common antigenic
determinants with lysosomal and endosomal membranes, including a 100 kD protein and proton pumps that may be involved in the acidification of the extracellular resorbing compartment. The basolateral membrane is highly enriched in
sodium pumps. Finally, the cytoplasm of the osteoclast is highly enriched in carbonic anhydrase, and bicarbonate-chloride exchange appears to regulate the intracellular pH of this cell. These observations are consistent with a scheme in
which, in the low pH environment of the bone-resorbing lacuna produced by the
osteoclast, the mineral phase dissolves, exposing the organic matrix to the action
of the secreted enzymes. The activity of these enzymes is in turn presumably
favored by the acidic milieu. All constituents of the matrix, whether mineral or
organic, then would be reduced to their elemental forms (ions and amino acids)
extracellularly. No phagocytic events would be required for the complete degradation of the bone matrix. According to these concepts, therefore, membrane iontransport mechanisms become the most important molecular aspect of bone resorption.
Resorption of the calcified extracellular bone matrix
by osteoclasts constitutes an essential step in the sequential process of bone remodeling (Frost, 1964;
Baron et al., 1984b). In carrying out this resorptive
function, osteoclasts are thereby involved in normal
bone turnover and growth (Kimmel and Jee, 1980;
Tran van et al., 1982a,b), in spontaneous or induced
tooth movement (Baron, 1973; Vignery and Baron,
19801,in bone-fracture healing (Frost, 19641, as well as
in a number of pathological resorptive processes such
as periodontal disease (Saffar and Baron, 1977; Saffar
and Makris, 19801, local and systemic resorptive effects
of cancers (Mundy and Spiro, 1981), Paget’s disease
and osteoporosis (Avioli and Krane, 1977).
Our laboratory has been studying the cellular and
molecular biology of the osteoclast and its function in
bone resorption. The goal of this brief review is to summarize our findings and concepts, based mostly on observations made in our own laboratory, but also on
relevant contributions of other investigators.
After describing the morphological characteristics of
this cell, its synthetic and secretory functions are dis0 1989 ALAN R. LISS, INC.
cussed. Both morphological and functional approaches
have led to the conclusion that the osteoclast is a
highly polarized cell. In this context, therefore, the issue of targeting secretory products to the ruffledborder apical domain, rather than to the basolateral
domain of the cell membrane, is discussed. Characterization of the apical compartment then is described in
terms of both its content and its limiting membrane,
i.e., the ruff led-border membrane. These studies also
demonstrate the existence of a distinct basolateral domain and establish the presence and functional importance of some ionic transport systems a t each pole of
the cell. The potential role of these systems then are
discussed inasmuch as they are directly related to the
function of the osteoclast, i.e., bone resorption.
Received September 16, 1988; accepted December 8, 1988.
Fig. 1. Osteoclasts along the bone surface in their resorbing lacunae.
Two osteoclasts, each with three nuclei in this plane of section, are
present within a resorbing site formed by two Howship’s lacunae; at
the light microscopic level, one can distinguish the basophilic cytoplasm, the sealing zones (arrows) limiting the highly vacuolated ruffled-border area (between arrows). Undecalcified section, Goldner
stain x 1,250. Modified from Baron and Vignery 1981, by copyright
permission of Pergamon Press.
The osteoclast is a giant multinucleated cell formed
by the asynchronous fusion of mononuclear hematopoietic precursors, most likely within the mononuclearphagocyte lineage (Jee and Nolan, 1963; Fishman and
Hay, 1962; Burger et al., 1982; KOand Bernard, 1981).
This cell is usually found singly or in low numbers at
one given time and site, characteristically at the interface between soft and calcified tissues in an area where
the bone matrix is fully mineralized (Fig. 1). Although
this interface includes the periosteum, most of the remodeling activity occurs along the endosteum, i.e., at
the interface between the calcified bone matrix and the
bone marrow. Osteoclasts and osteoblast-related cells
most probably are derived from hematopoietic and stroma1 stem cells, respectively, both of which are present
in bone marrow (Owen, 1978). At the light microscopic
(LM) level, the osteoclast is characterized by its size
(50-100 km in average), its multinucleation (usually
two to ten nuclei), and its presence within a resorptive
(Howship’s)lacuna along the edge of the calcified matrix-bone marrow interface. In good preparations, the
osteoclast is found in very close apposition to the calcified matrix; the area of the cell closest to the matrix
is characterized by densely stained patches at the periphery of the cell and a lightly stained, highly vacuolated, and striated center area corresponding to the
ruffled-border (Fig. 1,and see below). The cytoplasm is
usually strongly basophilic, granular, and foamy, with
vacuoles of varying sizes located mostly between the
nuclei and the ruffled-border area. The nuclei are characteristically heterogeneous in size, shape, and basophilia, a possible reflection of the asynchronous fusion
of mononuclear precursors (Miller et al., 1977). Ultrastructural analysis (Holtrop and King, 1977) of the os-
teoclast (Fig. 2) confirms and extends the LM observations. First, the morphological polarity of the cell is
evident, with a marked contrast existing between the
side facing the calcified matrix and the side of the cell
facing the bone marrow. Toward the bone surface, the
peripheral zone of the osteoclast is very closely apposed
to the extracellular matrix. This so-called “sealing
zone” (Schenk et al., 1967) is characterized by a very
narrow space (0.2 to 0.5 nm) between the plasma membrane of the cell and the calcified matrix and by the
presence of an organelle-free area in the adjacent cytoplasm, the “clear-zone” (Fig. 21, which is characteristically enriched in contractile proteins (King and
Holtrop, 1975; Marchisio et al., 1984). Toward the center of the cell-matrix interface, the plasma membrane
of the osteoclast manifests progressively deeper infoldings, which reach a high degree of geometric complexity. Numerous folds and ampullar spaces are present
which, due to the random obliquity of the sections, often appear as “vacuoles” in the cell’s cytoplasm. This
membrane has been suggested to be coated on its cytoplasmic side in a characteristic manner (Kallio et al.,
19711, but the exact nature of the coating material has
not yet been determined.
In all species tested so far (Fig. 2), one of the most
consistent morphological features of the cytoplasmic
organization of the osteoclast was the perinuclear distribution of multiple Golgi complexes, which entirely
surround each of the nuclei of the cell (Baron et al.,
1985b). We also recently found that all of these Golgi
stacks are functionally oriented with the trans-side
away from the nuclei (Baron et al., 1988a). In addition,
the osteoclast is characteristically rich in mitochondriae, free polysomes, and coated transport vesicles in
the Golgi areas, as well as in multiple vacuolar structures of heterogeneous size and shape, concentrated
mostly between the nuclei and the ruffled-border area.
As mentioned earlier, many of these vacuoles are pockets of extracellular space cut obliquely through the
folds of the plasma membrane. The osteoclast is not, as
often thought, poor in rough endoplasmic reticulum
(rER): this organelle is quite well developed, but it remains limited in extent relative to the large size of the
cell. The rER is usually concentrated in the basal portion of the cytoplasm of the osteoclast and in the perinuclear envelopes (Fig. 2).
Distribution of Acid Phosphatase and
Other Lysosomal Enzymes
It has been known for some time that one of the main
characteristics of the osteoclast is its enrichment in
acid phosphatase. This has been well demonstrated by
LM and electron microscopic (EM) studies (Fig. 2) and
by enzyme cytochemistry (Doty and Schofield, 1972;
Gothlin and Ericsson, 1971; Lucht, 1971; Baron et al.,
1985b, 1986b, 1988a). An isoenzyme of acid phosphatase that is resistant to incubation in the presence
of tartrate (tartrate-resistant acid phsophatase: TRAP)
seems to be the form in which the osteoclast is particularly (Minkin, 19821, if not specifically (Anderson and
Toverud, 19811, enriched. It is most important to emphasize that ultrastructural studies have shown that
this high concentration of lysosomal enzymes in the
osteoclast is not due to a high concentration of phago-
Fig. 2. General ultrastructural view of an osteoclast after localization of arylsulfatase. This micrograph shows an osteoclast attached to
the bone surface a t the sealing zones (SZ, arrows), which delimit the
ruffled border area (between arrows) and its complex membrane infoldings; large extracellular recesses (double arrows) are found deep
into the cell's cytoplasm and are close to the nuclei (n) and their
surrounding Golgi complexes; abundant mitochondria are found in
the osteoclast. Arylsulfatase is localized in all components of the biosynthetic pathway, including the endoplasmic reticulum (ER),the
numerous perinuclear Golgi complexes, and their associated vesicles,
which are often close to the infoldings of the ruffled-border membrane
(double arrows). Bar 1 km. x 9,000. Reproduced from Baron et al.,
1985, by copyright permission of the Rockefeller University Press.
cytic structures such as secondary lysosomes. Instead,
these enzymes are found, for the most part, in elements
of the exocytic pathway (Baron et al., 1985b, 1986b,
1988a). Consequently, this enrichment in lysosomal
enzymes does not reflect a high phagocytic activity, but
rather a high biosynthetic activity. Using a variety of
techniques to localize multiple enzymes, the presence
of these enzymes was demonstrated in the rER, in the
Golgi complex, and in numerous transport vesicles of
the osteoclast. Thus, localization of arylsulfatase and
beta-glycerophosphatase by enzyme cytochemistry and
localization of beta-glucuronidase and cathepsin C by
immunocytochemistry both have shown the abundant
concentration of these enzymes in the lumen of the rER
cisternae, including the perinuclear envelopes, in the
cisternae of the Golgi complexes and in very numerous
small (50 to 75 nm), coated vesicles in the Golgi complex areas and throughout the cytoplasm. These latter
are particularly abundant between the nuclei and the
deep portions of the ruffled-border membrane folds
(Baron et al., 198513, 1988a). In addition, some typical
secondary lysosomal vacuoles were found, which are
filled also with enzymes but are uncoated, are larger,
and have a heterogeneous content. These are, however,
relatively few and concentrated mostly in the basal
portion of the cell, facing the bone marrow compartment.
Evidence for Polarized Secretion of Newly Synthesized
Lysosomal Enzymes
Although strongly suggested by many studies (Vaes,
1968; Lucht, 1971; Doty and Schofield, 1972; Gothlin
and Ericsson, 1971), direct evidence for the secretion of
lysosomal enzymes by the osteoclast into the sealed-off
extracellular bone-resorbing compartment was, until
recently, still lacking. Localization of these enzymes by
enzyme cytochemistry has never provided convincing
results, in part because of artefacts of salt precipitation
that occur in the bone-resorbing lacuna (due most probably to the high concentration of calcium and phosphate in this specific microenvironment). It is also possible that an excess of endogenous substrate-to-enzyme
ratio in this compartment masks hydrolysis of exogenously added substrate and consequently prevents reliable localization. Therefore, an attempt was made to
use immunocytochemical methods to localize extracellular enzymes; recently one such enzyme, cathepsin C
(Mainferme et al., 1985), was found a t high and detectable concentrations in the subosteoclastic boneresorbing compartment (Baron et al., 1988a). In contrast, enzyme cytochemistry provided a better method
for the localization of lysosomal enzymes in intracellular organelles. Both methods agree, however, in showing high concentrations of these enzymes in the ER, all
cisternae of the Golgi complexes, and in most of the
small (50-75 nm), coated transport vesicles found in
the Golgi regions and in the cytoplasm (between the
perinuclear Golgi and the numerous folds and vacuoles
of the ruff led-border complex).
The overall conclusions from these studies are that
the osteoclast is actively engaged in the synthesis of
lysosomal enzymes that proceed through the Golgi and
are vectorially transported from the trans-Golgi region
to the ruff led-border apical membrane in coated transport vesicles. These transport vesicles then fuse with
the plasma membrane, exclusively at the ruffled border, and release their content into the bone-resorbing
compartment (Baron et al., 1988a). An attempt then
was made to determine how these enzymes are sorted
from other proteins and targeted to the apical membrane (Farquhar, 1985).
cellular organelle. Consequently, the possibility was
investigated that the osteoclast could lack such a
ligand-receptor system, or dispose of it differently
along the exocytic pathway to ensure the proper targeting of the enzymes to its apical membrane. It was
found that, indeed, the osteoclast expresses high concentrations of the cation-independent mannose-6 phosphate receptor (Baron et al., 1988a,b). The receptors
were found to be entirely codistributed with four different lysosomal enzymes along the exocytic pathway,
from their site of synthesis in the rER, through the
Golgi apparatus and the transport vesicles, to their delivery site at the ruffled-border membrane, where they
were present only at sites of recent budding of transport vesicles. These results were interpreted to indicate
that newly synthesized lysosomal enzymes in the osteoclast bear the mannose-6 phosphate recognition
marker and are targeted to the apical ruffled-border
membrane via their binding to the cation-independent
mannose-6 phosphate receptor in the Golgi apparatus.
The secretory enzymes are transported, bound to their
receptors, in small, coated vesicles from the trans-face
of the Golgi apparatus to the apical domain. These
transport vesicles fuse with the ruffled border membrane and release the enzymes upon reaching the boneresorbing compartment (Baron et al., 1985b, 1988a),
which is actively acidified by the osteoclast.
Evidence for a Low pH in the Bone-Resorbing
Compartment and for Active Acidification Process
by the Osteoclast
A number of previous studies suggested the possibility that the osteoclast might secrete acids during bone
resorption (Neuman et al., 1960; Vaes, 1968; Gay and
Mueller, 1974). These were, however, mostly indirect
observations, and the fact that collagenases, which
have a neutral pH optimum (Vaes, 1980), were considered to be the only enzymes capable of digesting collagen raised doubts as to whether or not the bone matrix
could be degraded in an acidic environment (Blair et
Role of the Mannose-6-PhosphateReceptor
al., 1986).
In most cells, lysosomal enzymes are sorted out of the
To address this issue, we developed a culture system
main flow of secretory and membrane proteins a t the in which, without isolation of the cells, active funclevel of the Golgi apparatus (Farquhar, 1985; Kornfeld, tional osteoclasts could be directly observed in situ
1986; Griffiths and Simons, 1986; von Figura and Ha- while resorbing a flat surface of bone (Baron et al.,
silik, 1986) and specifically targeted to late endosomes 1985b). When such flat fragments of bone were incualong the endocytic pathway (Brown et al., 1986). The bated in the presence of the weak base acridine orange,
newly synthesized enzymes are recognized by a specific large acidic compartments could be observed by epiflureceptor, the mannose-6-phosphate receptor (Creek orescence in close association with the osteoclasts. Beand Sly, 1984), which is highly concentrated in mem- cause such compartments disappeared upon isolation of
branes of the Golgi compartment (Brown and Farqu- the cells from their substratum, it was suggested that
har, 1984, 19871, and binds the mannose-6-phosphate the acidic compartment was indeed extracellular, i.e.,
recognition marker, which is added to the lysosomal in the subosteoclastic bone resorbing compartment
enzymes in the cis-Golgi elements. This mechanism en- (Baron et al., 1985b). More recently, this finding was
sures that the enzymes are sorted out of the flow of confirmed using an EM probe for low pH (Anderson et
other proteins synthesized by the cells. The enzymes al., 1984) and peroxidase-conjugated second antibodies.
remain bound to this mannose-6-phosphate receptor This probe accumulated in the subosteoclastic compartuntil they reach an acidified compartment where the ment and between the fingerlike folds of the ruffledlow pH induces their dissociation from the receptor. In border membrane, thereby demonstrating unequivothis manner, enzymes are released in the proper com- cally that this compartment is indeed acidic (Baron et
partment, i.e., the late endosome (Brown et al., 1986). al., 1985a).
However, the situation is quite different in the osTo determine if this was the result of osteoclastic
teoclast, because this cell secretes its newly synthe- activity, the cells were incubated in the presence of
sized enzymes instead of targeting them to an intra- ammonium chloride, thereby dissipating the existing
Fig. 3. Immunolocalization of the 100 kD membrane protein in an
osteoclast. The reaction product is present along the ruffled-border
membrane but is lacking at the sealing zone membrane (SZ); double
arrow indicates clear-cut limit between these two membrane domains. Note the ampullar dilations of extracellular space (ECS)at the
cytoplasmic end of the plasma membrane infoldings of the ruffled
border, which might appear in sections as intracellular vacuoles.
Small arrows show the reaction product exclusively at the luminal
side of the membrane. Bar, 600 nm. x 20,000. Reproduced from Baron
et al., 1985, by copyright permission of the Rockefeller University
pH gradient across the membrane. Upon reincubation
of the osteoclasts in normal culture medium, the cells
were able to reacidify the bone-resorbing compartment,
demonstrating that acidification is an active process
due to the osteoclast (Baron et al., 1985b).
Data from other laboratories have indeed supported
and confirmed these observations. First, fluid micropipetted from the osteoclast environment in vitro (a
method that does not, however, permit exact localization of the sample), suggested the presence of a low pH
(Fallon, 1984). Second, studies with isolated osteoclasts
placed on a slice of bone in vitro demonstrated that
these cells were more active in a slightly acidic culture
medium (Arnett and Dempster, 1986) and suggested
that under these conditions the cells more readily establish and maintain a pH gradient.
It is therefore reasonably well accepted now that the
osteoclast is able to acidify the bone-resorbing compartment formed at its apical surface, and this acidification
is the result of an active process performed by the cell
sosome. Therefore, the hypothesis was tested that the
ruff led-border membrane could share common antigenic determinants with lysosomal membranes. For
this purpose, a number of antibodies known to recognize lysosomal andor endosomal membrane antigens
(Baron et al., 1985a) were used, and immunocytochemical localization of these antigens in osteoclasts was
performed. The first and most striking result was the
presence of a 100 kD lysosomal membrane protein a t
the ruffled border of the osteoclast (Baron et al.,
1985b). Interestingly, this antigen was limited strictly
to the ruffled-border membrane (Fig. 3) but was not
present in detectable amounts at the sealing zone or
the basolateral membrane of the osteoclast. This observation thus established that 1)the osteoclast, although
not epithelial in nature, is able to establish and maintain distinct plasma membrane domains, i.e., polarized
apical and basolateral domains; and 2) the membrane
limiting the extracellular bone-resorbing compartment
shares some common antigen(s) with the limiting
membrane of lysosomes, thereby better establishing
the functional parallel between the bone-resorbing lacuna and secondary lysosomes. Further studies of
membrane proteins in the ruffled border area, however, indicated that some other lysosomal membrane
antigens were absent (Baron et al., 1985a), suggesting
some potentially important differences between these
two membranes.
Given the known role of a proton pump adenosine
Characterization of the Apical Plasma Membrane (Ruffled
Border) and of its Potential Role in Acidification
The apical bone-resorbing compartment, which is
characterized by a low pH, a high concentration of lysosomal enzymes, and the presence of the substrate for
these enzymes (the bone matrix proteins), is highly
reminiscent of the luminal content of a secondary ly-
triphosphatase (ATPase) in establishing and maintaining the pH gradient across the lysosomal membrane
(Ohkuma and Poole, 1978) and given the fact that most
ATPases are in the 100 kD molecular weight range,
Reggio et al. (1984), who first described this 100 kD
lysosomal membrane protein, tested the possibility
that it could be the protein transporting protons into
lysosomes and found that on immunoblots their antibody did not cross-react with the alpha subunit of the
sodiudpotassium or the calcium ATPase, both of
which are also in the 100 kD range; however, they
recognized the 100 kD proton/potassium ATPase purified from gastric mucosae. This strongly suggested, but
did not demonstrate, a possible link between the presence of this membrane protein in lysosomes (and in the
osteoclast ruffled border) and the fact that both membranes limit acidic compartments. According to these
observations, the proton-pump ATPase a t the ruff led
border could be of the gastric type. Some reports in the
literature have since indicated that the drug omeprazole, which specifically inhibits these pumps, decreased
bone resorption in organ cultures (Tuukkanen and
Vaananen, 1986) and the pH gradient formed by osteoclasts (Anderson et al., 1986). More recent data, although still preliminary, has more convincingly indicated that these pumps could be of the kidney type (i.e.,
electrogenic), inhibited by N-ethyl maleimide (NEM),a
compound known to inhibit specifically electrogenic
proton pumps from kidney or lysosomal membranes,
but not the (H + /K + )ATPase (Al-Awqati, 1986), and
are not inhibited by vanadate (Ghiselli et al., 19871, a
compound known to inhibit the protodpotassium ATPase. Preliminary data from our laboratory using isolated osteoclast membrane vesicles has also shown a
strong inhibition of acidification by NEM (Fuchs et al.,
unpublished data). In addition, the coating of the inner
surface of the ruff led-border membrane is strongly
reminiscent of the coating observed in kidney vesicles,
recently shown to be formed by crystalline arrays of
proton pump subunits (Brown et al., 1988).
All of these observations therefore strongly suggest
that the ruffled-border membrane is responsible for the
acidification of the bone-resorbing compartment via a
specific enrichment in proton pumps, most likely of the
NEM-sensitive electrogenic type.
Characterizationof the Basolateral Membrane and Its
Transport Mechanisms Potentially Associated
With Bone Resorption
Of all the plasma-membrane transport systems, two
are particularly important in the basolateral membranes of cells that transport protons across their apical membrane. These are the sodium pump (Na+,
K + IATPase, which often provides the primary force
for proton transport or associated cotransport (Cantley,
19811, and the bicarbonate-chloride exchanger, secreting the bicarbonate generated by the enzyme carbonic
anhydrase, which provides protons to the apical pumps
from COZ and water (Al-Awqati, 1978) and is highly
enriched in the osteoclast (Gay et al., 1974,1983;Vaananen and Parvinen, 1983).
Studies were therefore initiated that aimed at determining if the basolateral membrane of the osteoclast
was indeed enriched in these two transport molecules.
Using monoclonal antibodies t o both the alpha and
beta subunits of the (Na + ,K + )ATPase, immunocytochemistry showed the osteoclast to be highly enriched
in sodium pumps (Baron et al., 1986a). Not only was
the cell enriched, but this enrichment was SO pronounced that it became specific for the osteoclast relative to other cells present in sections of normal bone
tissue. In order to establish and quantitate this enrichment in sodium pumps further, we then proceeded to
measure the binding of radiolabeled ouabain, a specific
ligand for the alpha subunits of these pumps. About 5
million binding sites per osteoclast (Baron et al.,
1986a) were found, a relatively high number compared
with most other cells. Although this enrichment could
be related to functions other than bone resorption per
se, recent data from our laboratory indicates that the
activity of the (Na + , K + )ATPase is required for bone
resorption to occur. Thus, ouabain inhibits the release
of 45Ca from fetal rat long bones in culture as well as
the formation of resorption pits by isolated osteoclasts
(Prallet et al., 1988). As is the case for the proton pump
ATPase, our most recent data (Baron et al., 1988b)suggests that the sodium pump of the osteoclast is of the
kidney type (alpha 1) (Sweadner and Gilkeson, 1985)
and that it is concentrated mostly in the basolateral
domain of the plasma membrane.
To date there is no clear immunocytochemical or immunochemical data to demonstrate that the bicarbonate-chloride exchanger is present in the osteoclast.
However, recent transport data based on the measurement of intracellular pH in isolated cells indicates that
the osteoclast possesses such a system (Teti et al.,
1987). However, further work is necessary to characterize this transport mechanism both biochemically
and physiologically.
Cellular and Molecular Mechanisms of Bone Resorption
The various observations discussed above have led us
to reassess current views on the mechanisms by which
the multinucleated osteoclast performs its highly specialized function of resorbing bone, i.e. digesting an
extracellular calcified matrix. This review is concluded
by a summary of our current concepts on the molecular
aspects of osteoclast function.
The osteoclast is a multinucleated cell that is actively engaged in the synthesis, vectorial transport,
and polarized secretion of lysosomal enzymes toward
its apical pole. These secretory products are targeted to
the apical ruff led-border membrane by mechanisms
that involve cation-independent mannose-6-phosphate
receptors. These receptors bind to the mannose-6-phosphate recognition marker in the Golgi complex, and the
receptor-ligand complex, carried within small, coated
transport vesicles, dissociates upon reaching the low
pH established in the bone-resorbing compartment by
the osteoclast. The apical bone-resorbing compartment
is sealed off by the attachment of the osteoclast to the
calcified matrix and is actively acidified by the osteoclast. The plasma membrane of the cell is divided into
distinct domains. The apical membrane a t the ruffled
border shares common antigenic determinants with lysosomal and endosomal membranes, including a 100
kD protein and proton pumps. The basolateral membrane is highly enriched in sodium pumps. Both of
these membrane transport systems seem analogous to
the kidney forms rather than to the stomach and brain
forms, respectively. Finally, the cytoplasm of the osteoclast is highly enriched in carbonic anhydrase, and bicarbonate-chloride transport appear to regulate the intracellular pH of this cell.
Once the osteoclast forms a sealed-off bone-resorbing
compartment (subosteoclastic) and secretes both lysosomal enzymes and protons (producing a low pH environment), the mineral phase dissolves, exposing the
organic matrix to the proteolytic action of the enzymes.
Protons are provided to the proton pumps by the intracellular action of carbonic anhydrase and the generated bicarbonate is secreted basolaterally via a bicarbonate-chloride exchanger in the membrane. The
chloride exchanged in this process might be secreted
apically, in parallel with protons, via chloride channels. The calcium that would be generated in the resorbing compartment would be disposed of by transcytosis via calcium channels, calcium ATPases, or
sodium-calcium exchangers; the high basolateral concentration of sodium pumps suggests this latter as the
most likely mechanism (Krieger and Tashjian, 1980).
In this view of osteoclast function, all constituents of
the matrix, whether mineral or organic, would be reduced to their elemental forms (ions and amino acids)
extracellularly, and no intracellular phagocytic events
would be required for the complete degradation of the
bone matrix. According to these concepts, membrane
transport mechanisms become the most important molecular physiological aspect of bone resorption.
The present work was supported primarily by a
grant from the National Institutes of Health (DE04724). The author is very grateful to Mrs. Lynn Neff
for her expert technical help.
Al-Awqati, Q. 1978 H + transport in urinary epithelia. Am. J . Physiol., 235:F77-F88.
Al-Awqati, Q. 1986 Proton-translocating ATPases. Annu. Rev. Cell
Bid., 2:179-199.
Anderson, R.W., J.R.Falck, J.L. Goldstein, and M.S. Brown 1984 Visualization of acidic organelles in intact cells by electron microscopy. Proc. Natl. Acad. Sci. U.S.A., 81:4838-4842.
Anderson, T.R., and S.U. Toverud 1981 Further studies on the separation and identification of two phosphatases with acid optima
from rat bone. Calcif. Tissue Int., 33:261-267.
Anderson, R.E., D.M. Woodbury and W.S.S. Jee 1986 Humoral and
ionic regulation of osteoclast acidity. Calcif. Tissue Int.,
Arnett, T.R., and S.W. Dempster 1986 The effect of pH on bone resorption by rat osteoclasts in vitro. Endrocinology, 119:119-124.
Avioli, L.V., and S.M. Krane 1977 Metabolic Bone Disease. Academic
Press, New York.
Baron, R. 1973 Remaniement de la lame cribriforme et des fibres
desmodontales a u cours de la migration physiologique. J . Biol.
Buccale, 1:151.
Baron, R., S. Kellokumpu, L. Neff, S. Jamsa-Kellokumpu, A. Billecocq, B. Prallet, J. Beresford, J . Emanuel, K. Sweadner, and R.
Levenson 1988b Sodium pumps and bone resorption: presence of
basolateral kidney type alpha subunits in osteoclasts, effects of
ouabain on bone resorption and interactions with calcitonin in
MDCK cells. Calcif. Tissue Int., 42: (abstract).
Baron, R., and A. Vignery 1981 Behavior of osteooclasts during a
rapid change in their number induced by high doses of parathyroid hormone or calcitonine in intact rats. Metab. Bone Tis. Res.
Baron, R., A. Vignery, and M. Horowitz 1984 Lymphocytes, macro-
phages and the regulation of bone remodeling. In: Bone and Mineral Research 2. W.J. Peck ed., Elsevier, New York, pp. 175-243.
Baron, R.,L. Neff, J. Lippincott-Schwartz, D. Louvard, I. Mellman, A.
Helenius, and M. Marsh 1985a Distribution of lysosomal membrane proteins in the osteoclast and their relationship to acidic
compartments. J . Cell Biol., 101:53a (abstract).
Baron, R., L. Neff, D. Louvard, and J.P. Courtoy 19851, Cell-mediated
extracellular acidification and bone resorption: Evidence for a
low pH in resorbing lacunae and localisation of a 100-KD lysosomal membrane protein at the osteoclast ruffled border. J . Cell
Biol., 101:2210-2222.
Baron, R., L. Neff, C. Roy, A. Boisvert, and M. Caplan 1986a Evidence
for a high and specific concentration of (Na + ,K + )ATPase in the
plasma membrane of the osteoclast. Cell, 46:311-320.
Baron, R., L. Neff, P. Tran Van, J.R. Nefussi, and A. Vignery 1986b
Kinetic and cytochemical identification of osteoclast precursors
and their differentiation into multinucleated osteoclasts. Am. J .
Pathol., 122:363-378.
Baron, R., L. Neff, W. Brown, D. Louvard, P. Courtoy, and M.G. Farquhar 1988a Polarized secretion of lysosomal enzymes: co-distribution of cation independent mannose 6-phosphate receptors and
lysosomal enzymes in the osteoclast exocytic pathway. J . Cell
Biol., 106:1863-1872.
Blair, H.C., A.J. Kahn, E.C. Crouch, J.J. Jeffrey, and S.L. Teitelbaum
1986 Isolated osteoclasts resorb the organic and inorganic components of bone. J . Cell Biol., 102:1164-1172.
Brown, W.J., and M.G. Farquhar 1984 The mannose-6-phosphate receptor for lysosomal enzymes is concentrated in cis-Golgi cisternae. Cell, 36:295-307.
Brown W.J., and M.G. Farquhar 1987 The distribution of 215 kD
mannose 6-phosphate receptors within cis (heavy) and trans
(light) Golgi subfractions varies in different cell types. Proc. Natl.
Acad. Sci. U.S.A. (in press).
Brown, W.J., J . Goodhouse, and M.G. Farquhar 1986 Mannose6-Phosphate Receptors for Lysosornal Enzymes Cycle between the
Golgi Complex and Endosomes. J. Cell Biol., 103:1235-1247.
Brown, D., S. Gluck, and J. Hartwig 1987 Structure of the novel
coating material in proton-secreting epithelial cells and identification as a n H+ATPase. J. Cell Biol., 105~1637-1648.
Burger, E.H., J.W.M. Van der Meer, J.S. Van de Gevel, J.C. Gribnau,
C.W. Thesingh, and R. Van Furth 1982 In vitro formation of
osteoclasts from long term cultures of bone marrow mononuclear
phagocytes. J . Exp. Med., 156:1604-1634.
Cantley, L.C. 1981 Structure and mechanism of the ( N a + ,
K + IATPase. Curr. Top. Bioenerg. 11:201-237.
Creek, K.E., and W.S. Sly 1984 The role of the phosphomannosyl
receptor in the transport of acid hydrolases to lysosomes. In: Lysosomes in Biology and Pathology. J.T. Dingle, R.T. Dean, and
W.S. Sly, eds. Elsevier, Amsterdam, pp. 63-82.
Doty, S.B., and B.H. Schofield. 1972 Electron microscopic localization
of hydrolytic enzymes in osteoclasts. Histochem. J., 4:245-258.
Fallon, M.D. 1984 Bone resorbing fluid from osteoclasts is acidic-An
in vitro micropuncture study. In: Endocrine Control of Bone and
Calcium Metabolism. Cohn D.V. et al. eds. Amsterdam, Elsevier,
p. 144.
Farquhar, M.G. 1985 Progress in unraveling pathways of Golgi traffic. Annu. Rev. Cell Biol., 1:447-88.
Fishman, D.A., and E. Hay 1962 Origin of the osteoclast from regenerating newt limbs. Anat. Rec. 143:329-334.
Frost, H.M. 1964 Mathematical Elements of Bone Remodeling.
Charles C. Thomas, Springfield, Illinois.
Gay, C.V., and W.J. Mueller 1974 Carbonic-anhydrase and osteoclasts: localization by labeled inhibitor autoradiography. Science,
Gay, C.V., M.B. Ito, and H. Schraer 1983 Carbonic anhydrase activity
in isolated osteoclasts. Metab. Bone Dis. Rel. Res., 5:33-39.
Ghiselli, R., H. Blair, S. Teitelbaum, and S. Gluck 1987 Identification
of the osteoclast proton pump. J. Bone Min. Res. 2:Suppl. 1
Gothlin, G., and J.L.E. Ericsson 1971 Fine structural localization of
acid phosphomonosterase in the brush border region of osteoclasts. Histochemie, 28:337-344.
Griffiths, G., and K. Simmons 1986 The trans Golgi Network Sorting
at the Exit Site of the Golgi Complex. Science, 234:438-443.
Holtrop, M.E., and G.J. King 1977 The ultrastructureofthe osteoclast
and its functional implications. Clin. Orhop. Rel. Res., 123:
Jee, W.S.S., and P.D. Nolan 1963 Origin of osteoclasts from the fusion
of phagocytes. Nature, 200:225-226.
Kallio, D.M., P.R. Garant, and C. Minkin 1971 Evidence of coated
membranes in the ruffled border of the osteoclast. J . Ultrastruct.
Res., 37:169-177.
Kimmel, D., and W.S.S. Jee 1980 A quantitative histologic analysis of
the growing long bone metaphysis. Calcif. Tissue Int. 32:
King, G.J., and M.E. Holtrop 1975 Actin-like filaments in bone cells
of cultured mouse calvaria as demonstrated by binding to heavy
meromyosin. J . Cell Biol., 66:445-451.
KO,J.S., and G.W. Bernard 1981 Osteoclast formation in vitro from
bone marrow mononuclear cells in osteoclast-free bone. Am. J.
Anat., 161:415 -425.
Kornfeld, S. 1986 Trafficking of lysosomal enzymes in normal and
disease states. J. Clin. Invest., 77:l-6.
Krieger, N.S., and A.H. Tashjian 1980 Parathyroid hormone stimulates bone resorption via a Na-Ca exchange mechanism. Nature,
Lucht, U. 1971 Acid phosphatase of osteoclasts demonstrated by electron microscopic histochemistry. Histochemie, 28:103-117.
Mainferme, F., R. Wattiaux, and K. von Figura 1985 Synthesis, transport and processing of cathepsin C in Morris hepatoma 7777 cells
and rat hepatocytes. Eur. J. Biochem., 153:211-216.
Marchisio, P.C., D. Cirillo, L. Naldini, M.V. Primavera, A. Teti, and A.
Zambonin-Zallone 1984 Cell-substratum interaction of cultured
avian osteoclasts is mediated by specific adhesion structures. J.
Cell Biol., 99:1696-1705.
Miller S., W.S.S. Jee, D.B. Kimmel, and L. Woodbury 1977 EHDP
effects on incorporation and accumulation of osteoclast nuclei.
Calcif. Tissue Res., 22:243-252.
Minkin, C. 1982 Bone acid phosphatase: Tartrate resistant acid phosphatase as a marker of osteoclast function. Calcif. Tissue Int.,
Mundy, G., and T.P. Spiro 1981 The mechanisms of bone metastasis
and bone destruction by tumor cells. In: Bone Metastases. L.
Weiss and H.A. Gilbert, eds. G.K. Hall, Boston, pp. 64-82.
Neuman, W.F., B.J. Mulryan, and G.R. Martin 1960 A chemical view
of osteoclasts based on studies with Yttrium. Clin. Orhop.,
Ohkuma, S., and B. Poole 1978 Fluorescence probe measurement of
the intralysosomal pH in living cells and the perturbation of pH
by various agents. Proc. Natl. Acad. Sci. U.S.A., 75:3327-3331.
Owen, M. 1978 Histogenesis of bone cells. Calcif. Tissue Res.,
Prallet, B., J. Beresford, L. Neff, and R. Baron 1988 Inhibition of
sodium pumps by ouabain decreases bone resorption in fetal rat
long bones and isolated rat osteoclast cultures. J. Bone Min. Res.,
3:Suppl. 1, (abstract).
Reggio, H., D. Bainton, E. Harms, E. Coudrier, and D. Louvard 1984
Antibodies against lysosomal membranes reveal a 100 kD protein
which cross-reacts with purified H + , K + ATPase from gastric
mucosa, J . Cell Biol., 99:1511.
Saffar, J.L., and R. Baron 1977 A quantitative study of osteoclastic
bone resorption during experimental periodontal disease in the
golden hamster. J . Periodont. Res., 12:387-394.
Saffar, J.L., and G.P. Makris 1986 A morphological and quantitative
study of osteoclast changes during the progress of periodontitis in
the hamster. J. Biol. Buccale, 14:255-262.
Schenk, R., D. Spiro, and J. Wiener 1967 Cartilage resorption in tibia1
epiphyseal plate of growing rats. J. Cell Biol., 34:275-291.
Sweadner, K.J., and R.C. Gilkeson 1985 Two isozymes of the
(Na+ ,K + )ATPase have distinct antigenic determinants. J. Biol.
Chem., 260:9016-9022.
Teti, A,, H. Blair, A. Kahn, J. Konsek, C. Koziol, A. Zambonin-Zallone. S. Teitelbaum. and P. Schlesinaer 1987 Intracellular PH
regu'lation of isolated osteoclasts b y chloridefbicarbonate exchange. J. Bone Min. Res. 2:Suppl. 1 (abstract).
Tran Van, P., A. Vignery, and R. Baron 1982a An electron microscopic
study of the bone remodeling sequence in the rat. Cell Tiss. Res.,
Tran Van, P., A. Vignery, and R. Baron 19821, Cellular kinetics of the
bone remodeling sequence in the rat, Anat. Rec., 202:445.
Tuukkanen, J., and H.K. Vaananen 1986 Omeprazole, a specific inhibitor of H + -K + ATPase, inhibits bone resorption in vitro. Calcif. Tissue Int., 38:123-125.
Vaananen, H.K., and E.K. Parvinen 1983 High active isoenzyme of
carbonic anhydrase in rat calvaria osteoclasts. Histochemistry,
Vaes, G. 1968 On the mechanisms of bone resorption: the action of
parathyroid hormone on the excretion and synthesis of lysosomal
enzymes and on the extracellular release of acid by bone cells. J .
Cell Biol., 39:676-697.
Vaes, A. 1980 Collagenase, lysosomes, and osteoclastic bone resorption. In: Collagenase in Normal and Pathological Connective Tissues. D.E. Woolley and J.M. Evanson, eds. J. Wiley & Sons, London. pp. 185-207.
Vignery, A., and R. Baron 1980 Dynamic histomorphometry of alveolar bone remodeling in the adult rat, Anat. Rec., 196:191.
von Figura, K., and A. Hasilik 1986 Lysosomal enzymes and their
receptors. Annu. Rev. Biochem., 55:167-193.
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