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MICROSCOPY RESEARCH AND TECHNIQUE 33:165-170 (1996)
Role of Microscopy in Elucidating the Mechanism and
Regulation of the Osteoclast Resorptive Apparatus
CAROL V . GAY
Department of Biochemistry and Molecular Biology and Department of Poultry Science, The Pennsylvania State University,
University Park, Pennsylvania 16802
KEY WORDS
Osteoclasts, Bone resorption, Carbonic anhydrase, Proton-translocating ATPase
INTRODUCTION
Microscopy has spearheaded investigations into understanding cell function in bone, perhaps more than
for any other tissue. The reason is that bone cells lie
within or on the extracellular substance of bone, a material not unlike reinforced concrete. It is the purpose
of this paper' to survey the role microscopy has played
in elucidating osteoclast function.
Osteoclasts were identified nearly a century and a
half ago by microscopists (Kolliker, 1873; Robin, 1849).
These early morphologists correctly interpreted that
osteoclasts are bone resorbing cells. This conclusion
was based on the crumbling appearance of bone beneath osteoclasts and the fact that pieces of bone appeared to be ingested by the cells. Modern scanning
electron microscope studies have made it imminently
clear that osteoclasts degrade bone by making resorption pits, whereas other cells such as monocytes, macrophages, and certain tumor cells that resorb devitalized bone do not make resorption pits in vivo (Jones et
al., 1985). As microscopic techniques such as immunocytochemistry, histochemical reaction capture methods, autoradiography, and in situ hybridization methods were developed, insight into the cellular basis of
bone resorption has progressed steadily.
The task of an osteoclast is defined by the composition of extracellular bone matrix, which is mainly hydroxyapatite crystals packed into parallel arrays of a
fibrous protein, type I collagen. Two eroding systems,
then, are needed: a way to dissolve mineral and a
means of degrading protein. The former requirement is
now well established as an acid-secreting mechanism
that involves carbonic anhydrase and a vacuolar-type
H+-ATPase; the latter is due to proteolytic enzymes.
The carbonic anhydrases are central to COOmetabolism, and the range of tissues served by this family of
enzymes is consequently widespread. That many investigators have become intrigued by carbonic anhydrase
is evident from the recent exhaustive publication of
Dodgson et al. (1991). Not only is carbonic anhydrase
notable for its rapid turnover rate, but it also subserves
an array of physiological processes by epithelia. These
processes include gas exchange a t the blood lung interface, elaboration of a bicarbonate rich fluid by tissues
such as pancreas and salivary glands, and acidification
of luminal fluids by the proximal renal tubule and the
gastric mucosa. A number of isoforms have been identified, such as one for muscle cells and another for mitochondria.
The important carbonic anhydrase isoform in osteoclasts is CAII. This has been shown by immunocytochemistry for chicken (Gay et al., 1974), rat (Vaananen
and Parvinen, 1983), and human (Vaananen, 1984) osteoclasts. A CAI, deficiency syndrome in humans is associated with a mild form of osteopetrosis (Sly et al.,
19831, a finding that suggests a role for carbonic anhydrase in osteoclastic bone resorption.
Messenger RNA for CAI1 has been localized by in situ
hybridization, a molecular tool that exploits the power
of microscopy to capture space relationships, in osteoclast-like cells derived from an osteosarcoma (Zheng et
al., 1993) as well as in neonate rat osteoclasts (Laitala
and Vaananen, 1993). Both studies found good corroboration of in situ hybridization with Northern analysis.
The latter study also showed that active osteoclasts
expressed substantial levels of CAI1 mRNA, whereas
nonresorbing osteoclasts expressed very little mRNA.
Transcription of CAI1 mRNA has been shown to be regulated by 1,25-dihydroxyvitamin D, in osteoclast precursor cells (Billecocq et al., 1990; Lomri and Baron,
MECHANISM OF MINERAL DISSOLUTION
1992).
Localization and Role of Carbonic Anhydrase
The carbonic anhydrase isoform CAVhas been localCarbonic anhydrase reversibly hydrates COz to form ized in the matrix of osteoclast, renal, and gastric micarbonic acid. Definitive identification of carbonic an- tochondria by ultrastructural immunocytochemistry,
hydrase in osteoclasts came from imrnunocytochemi- but the role of CAv is not currently understood (Karcal, autoradiographic, and histochemical microscopic hukorpi et al., 1992).
Much of CAI1 is distributed throughout the cytoanalyses. Using these methods, carbonic anhydrase
has been localized in osteoclast cytoplasm of chickens plasm in osteoclasts. The role of cytosolic carbonic an(Anderson et al., 1982; Gay and Mueller, 1974), rats
(Marie and Hott, 1987; Simasaki and Yagi, 1960;
Sundquist et al., 1987; Vaananen and Parvinen,
1983)), mice (Jilka et al., 19851, and humans (VaanaReceived Januarv 3. 1994: accepted August 18. 1994.
nen, 1984).Its role in osteoclasts has been reviewed by
Address reprint requests to Carol V. Gay, 468A N. Frear Building, University
Gay (1991, 1992).
Park. PA 16802.
0 1996 WILEY-LISS, INC.
166
C.V.GAY
hydrase is not completely understood; however, a potential role is to facilitate diffusion of CO, from the cell
interior to the cell surface, as described in model systems by Enns (1967) and Shultz (1980).Given the enormity of osteoclasts, which can have a volume 50 times
or more than other cells, facilitated diffusion is likely
to be involved.
It was observed by ultrastructural immunocytochemistry that carbonic anhydrase in osteoclasts is,
under some circumstances, associated with the inner
surface of the ruffled border (Anderson et al., 1982; Cao
and Gay, 1985). Membrane-associated carbonic anhydrase has also been reported in a giant cell tumor of
bone (Kuwahara et al., 1986). The observations of
membrane-associated carbonic anhydrase led us to postulate that one role of carbonic anhydrase is to provide
hydrogen ions to a proton-pumping ATPase in the ruffled border of osteoclasts.
Localization of the Proton-Translocating ATPase
One way to localize an enzyme is by capturing histochemical reaction products. In 1985, a number of
methods existed for localizing Na+,K+-ATPases and
Mf+ +,Ca++-ATPases, however, none existed for an
H -ATPase at the ultrastructural level. Using the existing histochemical methods for ATPases as models,
we devised a way to identify sites of H+-ATPase activity (Akisaka and Gay, 1986). The reaction depended on
the presence of ATP and Mg++ a t high pH. Released
phosphates were trapped in the conventional manner
by lead ions. Staining, which was observed along the
ruffled border and in cytoplasmic vesicles, was blocked
by proton-pumping ATPase inhibitors p-chloromercuribenzoate, tri-n-butyltin, and duramycin. Meanwhile, by use of an antibody to gastric H+,K+-ATPase
immunostaining of the ruff led border was demonstrated (Baron et al., 1985). In 1989, antibodies to bovine kidney vacuolar H+-ATPase were found t o bind
specifically to the ruffled border (Blair et al., 1989). In
addition, Vaananen et al. (1990) used antibodies to
Neurospora crassa vacuolar ATPase and found ruffled
borders of both rat and chicken osteoclasts to be immunostained. It is now known that the osteoclast H+-ATPase is not of the gastric type. Presumably, epitopes in
common to both types of ATPase antibodies (gastric
and vacuolar) recognized the antigen. Thus, microscopic methods, both histochemical and immunochemical, have revealed sites of a proton ATPase in the ruffled border where it was hypothesized to be.
An intense investigation to demonstrate function of
the proton ATPase has followed. These studies have
made it clear that pumping of protons to the exterior of
osteoclasts is driven by a vacuolar type pump (Bekker
and Gay, 1990; Blair et al., 1989, 1991; Chatterjee et
al., 1992; Vaananen et al., 1990). Regulation of expression of the H+-ATPase was the next logical quest, and
here microscopic methods also played a role. In situ
hybridization has allowed localization of mRNA transcripts for the vacuolar H+-ATPase in isolated osteoclasts from neonate rats (Laitala and Vaananen, 1993).
Additionally, substantial levels of mRNA were present
in the cytoplasm. Northern analysis revealed high levels of transcript in active osteoclasts and markedly re-
Fig. 1. Diagram summarizing the mechanisms of osteoclast acidification and their potential regulation by parathyroid hormone
(PTH), calcitonin (CT), and 17P-estradiol (EJ. Acidification depends
on carbonic anhydrase (CA) activity and a proton-translocating
ATPase and is enhanced ( + ) by ETH and diminished (-) by CT and
E,. At least one signaling pathway utilized by PTH appears to involve
CAMP.See text for discussion and references.
duced levels in inactive cells. Amplified expression of
the H+-ATPase has been shown in late stage osteoclasts precursors (Kurihara et al., 1990; Wang et al.,
1993).
Insights Into Osteoclast Acidification Using
Fluorescence Microscopy
A microscope photometer and a pH-sensitive dye,
acridine orange, have been used to monitor changes in
acid levels in single living osteoclasts. Acridine orange
behaves as a weak base that diffuses through intact
cell membranes but is sequestered when protonated in
acidic compartments (Moriyama et al., 1982). Thus, increased levels of fluorescence are proportional to
trapped protons. Cells were first neutralized with 20
mM ammonium chloride added to culture medium;
then, by removal of the neutralizing solution, acidification was reinitiated (Hunter et al., 1988, 1991). The
sites of trapped acid appeared to be within intracellular
vesicles. It is believed that the vesicles fuse with the
ruffled border membrane, releasing both acid and proteolytic enzymes into the resorption lacuna. This view
is supported by electron microscopic observations that
have revealed many vesicles in inactive osteoclasts but
few in active cells (for reviews see Gay, 1992; Holtrop,
1991). Further, a unique histochemical marker, an alkaline p-nitrophenyl phosphatase, has been found to
stain both vesicles and ruff led border membrane but
not other membranes of the osteoclast (Fukushima et
al., 1991). The fusion of vesicles with the ruffled border
membrane is believed to be the means of positioning
the proton pump in the ruffled border (Fig. 1).
Table 1 summarizes and Figure 1 diagrams the ef-
MICROSCOPY AND OSTEOCLAST FUNCTION
TABLE 1 . Detection of changes in protonated acridine omnge by
fluorescence microscopy of isolated osteoclasts treated with
regulatory substances'
Treatment
A. Calcitonin (7 x lo-" M)
Acetazolamide
M)
HTS (10-9 MI
B. PTH (lo-' M)
DbcAMP
M)
PTH (lo-' M)
PTH + alloxan (lo-' M)
Cholera toxin (0.01 &g/ml)
C. Estradiol (2 x lo-'' M)
Diethylstilbestrol
( x 10-lOM)
Estradiol (2 x lo-' M) +
PTH
M)
Estradiol (2 x lo-* M) +
DbcAMP
M)
Estradiol (2 x lo-' M) +
alloxan (lo-' M)
Effects on
acidification'
(% change
t s.d.)
1 (44 f 4)
1 (30 f 3)
1 (48 t 6)
t (24 f 4)
t (45 ? 4)
t (27 f 5)
1 (20 f 5)
t (38 * 6)
.1 (41 f 3)
.1 (39 f 2)
Reference
Gay et al. (1993)
Hunter et al. (1988)
Hunter et al. (1991)
Gay et al. (1993)
May et al. (1993)
Gay et al. (1993)
1 (27 f 3)
1 (45 * 2)
1 (47 t 3)
'Abbreviations: DbcAMP, dibutyryl cyclic adenosine monophosphate;HTS, 543hydroxybenzoyllthiophene-2-sulfonamide;FTH, parathyroid hormone.
'Based on single cell values averaged for 20 cells per data point at 1 h treatment
times.
fects of some regulatory agents on osteoclast acid production. Both calcitonin and the carbonic anhydrase
inhibitors, acetazolamide and hydroxythiophene sulfonamide, blocked reacidification of acridine orange
stained osteoclasts (Table 1A). These results substantiate that carbonic anhydrase plays an integral role in
the acidification mechanism. Furthermore, since calcitonin when injected into chickens reduced the association of carbonic anhydrase along the inner face of the
ruffled border (Anderson et al., 19821,it is possible that
a role of calcitonin may be to control the supply of
hydrogen ions to the proton pump by altering the position of carbonic anhydrase. However, the physiological role of calcitonin is unclear. Its effects in humans
appear to be transient and of little use in controlling
trabecular bone volume or calcium homeostasis
(Mundy, 1990).
Parathyroid hormone (PTH) was found to enhance
acidification (Table 1B). Dibutyryl cAMP mimicked
the PTH effect, implying that the PTH response is
CAMP-dependent. Inhibition of CAMP generation by alloxan, an inhibitor of adenylate cyclase (Cohen and
Bitensky, 19691, blocked the PTH response. The involvement of a G-protein of the Gs type, whose major
role is adenylate cyclase stimulation, was shown by the
use of cholera toxin, a substance which permanently
activates the a-subunit of Gs-proteins. In addition, pertussis toxin which prevents receptor interaction with
G-proteins other than Gs-protein blocked the PTH
stimulatory effect on acid production (May and Gay,
1993). The implication of this is that PTH interacts
with more than one type of G-protein. These results
suggest that PTH regulates osteoclast acidification
through at least two signaling pathways. However,
these inhibitor studies require confirmation by other
approaches due to the potential pitfalls associated with
167
the use of inhibitors on living cells. In an earlier study,
PTH was found to stimulate CAMP levels in isolated
osteoclasts (It0 et al., 1985). Carbonic anhydrase has
been identified as an effector molecule for the PTHcAMP signaling pathway (Dietsch, 1987; Silverton et
al., 1987). It is possible that these actions of PTH occur
as a result of direct activation on osteoclasts. The isolated osteoclasts are relatively pure preparations and
typically include less than 10% of other cell types, only
2% of which are alkaline phosphatase positive (Gay et
al., 1983,1993). Secondly, PTH has been shown to bind
specifically to osteoclasts, as will be discussed in the
next section. However, proof of direct activation of osteoclasts by FTH awaits the demonstration of receptors
at protein and mRNA levels.
Also, as shown in Table lC, 17P-estradiol and diethylstilbestrol were found to slow reacidification of osteoclasts. The stimulatory effects of PTH and its second
messenger cAMP were overridden by treatment with
estrogen. This implies that the action of the estrogens
is to inhibit a step in the signal transduction pathway
at a later point than cAMP production.
By the use of culture methods, a fluorescent probe,
and a photometer integrated into a microscope it has
been possible to gain insight into one function of individual living osteoclasts: the process of acidification. It
should be pointed out that the use of photometry lacks
the precision that can be obtained by the use of instrumentation capable of processing ratiometric images.
The use of acridine orange to show acidification in lysosomes, as well as its several other uses, is reviewed
by Zelenin (1993).
Detection of Specific Binding of Calciotrophic
Hormones to Osteoclast Surfaces
Parathyroid Hormone. Results obtained from
studying the effects of parathyroid hormone on acidification by osteoclasts in relatively pure cultures of osteoclasts (Table 1A) led us to devise a way to test
whether or not parathyroid hormone could bind specifically to osteoclasts (Agarwala and Gay, 1992). Osteoclasts were isolated from 2-3-week-old chick tibia and
maintained in culture 4-6 days. A technique which
had been devised to show specific PTH binding to kidney cell surfaces (Niendorf et al., 1988) was employed.
Biotinylated bovine PTH was applied to the cultured
osteoclasts for 2-20 min. After rinsing away unbound
biotinylated PTH, avidin labeled with fluorescein
isothiocyanate was applied and the excess removed by
rinsing. Bright spots of fluorescence were observed on
the osteoclast surfaces. The surface binding displayed
the properties of receptor-dependent binding, namely
saturability, time dependence, temperature dependence, and competition with unlabeled PTH. In the
same study, osteoblasts were found to be intensely
stained, in accordance with other studies. PTH binding
to osteoclasts has also been shown by well-controlled
immunocytochemistry in sections of rat bone (Rao et
al., 1983) and 1251-PTHlabeling of isolated avian osteoclasts by autoradiography (Teti et al., 1991).
The amount of PTH that binds to osteoclasts is substantially less than that which binds to osteoblasts
(Agarwala and Gay, 1992). Further, osteoclast surfaces
168
C.V. GAY
are rapidly cleared of bound PTH (within 20 min).
These characteristics may contribute to the difficulty
in showing the direct involvement of PTH with osteoclasts. The demonstration of specifially bound PTH to
osteoclasts supports the view that osteoclasts are directly regulated by PTH, in addition to indirect regulation through the action of PTH on osteoblasts. However, the magnitude of both direct and indirect effects
of PTH on osteoclasts has not been assessed.
Calcitonin. The binding of radiolabeled calcitonin
to osteoclasts was first demonstrated in rat bone by
Warshawsky et al. (1980), who used autoradiography
at the ultrastructural level. Rao and colleagues (1981)
have shown binding of endogenous calcitonin to osteoclasts in sections of rat bone using antibodies to calcitonin. Application of radiolabeled calcitonin to isolated
osteoclasts followed by autoradiography has shown substantial binding of calcitonin to rat osteoclasts, while
binding to avian osteoclasts was undetectable (Nicholson et al., 1986, 1987). This has led to the view that
avian and mammalian osteoclasts are regulated by substantially different processes. However, it is well documented that avian osteoclasts respond to calcitonin in
low doses (for a review, see Gay, 19881, providing evidence for the presence of calcitonin receptors in avian
osteoclasts. Apparently, rat osteoclasts have substantially more calcitonin receptors than the avian cells.
By using the biotinylated hormone technique of
Niendorf et al., 1988 and Agarwala and Gay, 1992,
specific binding of calcitonin to avian osteoclasts has
now been shown (Hall et al., 1994). Living osteoclasts
were exposed to biotinylated calcitonin at room temperature. The cells were then chilled to disrupt microtubules and prevent possible uptake of the labeled hormone. Bound biotinylated hormone was made visible
by using fluorescein-tagged avidin. In order to amplify
the signal, a biotinylated antibody to avidin was applied, followed by a second application of fluorescein
tagged avidin. Characteristics of receptor binding were
shown: saturability, time dependence, competition
with unlabeled calcitonin and lack of competition by
unrelated peptides. Clearance from the cell surface occurred in 10 min, even more rapidly than found for
PTH. Because avian osteoclasts resorb bone more aggressively than osteoclasts derived from other species
(Jones et al., 1986), it is reasonable to speculate that
the avian cell is more tightly regulated and utilizes
fewer receptors.
Estradiol. Nuclear estrogen receptors have been
described in osteoblasts (Turner et al., 19931, osteoblast-like cells (Eriksen et al., 1988; Komm et al.,
19881, and osteoclasts (Oursler et al., 1991). However,
the response of osteoclasts to 17P-estradiol in the acidine orange assay used to monitor acidification (Table
1C) was detectable within 10-15 min. The rapidity of
this response suggested that estrogen may also be acting at a nonnuclear site. Further, proton pumping by a
vesicle preparation of osteoclast plasma membranes
was inhibited by 17p-estradiol at 2 x lop8 M (Gay et
al., 1993).
In order to further evaluate possible interaction of
estrogen a t plasma membrane, a complex of 17P-estradiol conjugated to bovine serum albumin-fluorescein
isothiocyanate has been applied t o osteoclast surfaces
(Brubaker and Gay, 1994). Binding was observed with
fluorescence microscopy; it was dose- and time-dependent and could be blocked with 17P-estradiol or tamoxifen, an estrogen antagonist. Both 17P-estradiol and
the 17p-estradiol-BSA-FITC complex stimulated cell
shape changes within minutes of application, further
documenting rapid responses that point to the existence of nonnuclear receptors.
How the estrogens affect osteoclasts in vivo is unclear. For example, they have failed to directly influence bone in organ cultures (Caputo et al., 1975). On
the other hand, the importance of estrogens in the reduction of bone loss in the immediate postmenopausal
period is well known (Lindsay et al., 1976, 1980).
MECHANISM OF ORGANIC
MATRIX DEGRADATION
Lysosomal proteinases are secreted into the resorption pit in a manner that is regulated by parathyroid
hormone, calcitonin, and other hormones (see review
by Delaissb and Vaes [19921).The most important proteinases appear to be cathepsin B and L, members of
the cysteine proteinase family, which can ef€iciently
degrade collagen a t an acidic pH. Most studies on matrix degradation have involved biochemical analyses.
However, the spatial precision afforded by microscopy
has shown that osteoclasts synthesize B, L and D cathepsins on the basis of immunolocalization and that the B
and L forms are secreted, whereas D cathepsin remains
as an intracellular enzyme (Goto et al., 1992,1993).An
ultrastructural study has shown that matrix is not destroyed when bone explants have been cultured in the
presence of the cysteine proteinase inhibitor, leupeptin
(Everts et al., 1988).
The question of the presence and secretion of procollagenase by osteoclasts is not completely resolved.
The major source of collagenase in bone is osteoblasts
(Vaes et al., 19921, although immunoreactive collagenase in osteoclasts has not been reported (Delaisse and
Vaes, 1992), suggesting that collagenase found in resorption lacunae may be secreted by osteoclasts. Further support for this view is provided by the detection
of collagenase mRNA by in situ hybridization in odontoclasts (Okamura et al., 1993). The role of collagenase
in resorption pits provides an intriguing problem since
collagenase is inactive at a low pH. An attractive hypothesis presented by Delaisse and Vaes (1992) poses
the possibility that collagenase is activated in regions
of the pit that have been neutralized by the HPO,'
released as hydroxyapatite is dissolved.
SUMMARY
Microscopic studies have assisted in revealing some
of the components of the resorptive apparatus of osteoclasts, specifically carbonic anhydrase and the protontranslocating ATPase. Further, microscopy has helped
substantiate the types of proteolytic enzymes secreted
into the resorption lacuna. Regulatory agents affecting
the resorptive process in vitro include parathyroid hormone, 17P-estradio1,calcitonin, and 1,25-dihydroxyvitamin D,. Studies showing the specific binding of parathyroid hormone, estradiol, and calcitonin to osteoclast
MICROSCOPY AND OSTEOCLAST FUNCTION
plasma membrane are discussed. While specific binding suggests that direct effects may occur, further investigation is needed to substantiate this possibility.
ACKNOWLEDGMENTS
The osteoclast acidification studies were supported
by NIH grant DE 04345.
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