THE EFFECTS OF PARATHYROID HORMONE, COLCHICINE, AND CALCITONIN ON THE ULTRASTRUCTURE AND THE ACTIVITY OF OSTEOCLASTS IN ORGAN CULTURE

The ultrastructure of osteoclasts was examined in fetal rat bones after stimulation or inhibition of resorption in culture. A central ruffled border area completely encircled by a clear zone was considered to represent the resorbing system of the cell. The proportion of ruffled border and clear zone in osteoclast cross sections was compared with changes in bone resorption as measured by the release of previously incorporated radioactive calcium (⁴⁵Ca). In control cultures 55% of the osteoclast cross sections showed an area closely apposed to bone and this consisted mainly of clear zone; only 11% showed ruffled borders. Treatment with parathyroid hormone (PTH) increased ⁴⁵Ca release, increased the frequency of finding areas closely apposed to bone (79%), and markedly increased the frequency of the ruffled border area (64%). Colchicine given concurrently with PTH decreased the number of osteoclasts. Colchicine or calcitonin treatment after PTH stimulation decreased the proportion of ruffled border area significantly by 1 h; this was followed by a decrease in ⁴⁵Ca release. These inhibited osteoclasts resembled osteoclasts from control, unstimulated cultures, suggesting that the cells had returned to their inactive state. Colchicine-treated osteoclasts also showed a loss of microtubules and a massive accumulation of 100 Å filaments, suggesting that synthesis of microtubular subunits had increased.     

Osteoclasts and their relationship to bone as studied by electron microscopy

Summary Osteoclasts in metaphyses from young rats were systematically sectioned at different levels. Two types of osteoclasts were recognized. One type had no ruffled border while the other, and predominant type contained a ruffled border in a part of its length; some of the latter contained two ruffled borders. The closest contact between osteoclast and bone occurred at the level of the ruffled border and this bone under the border showed characteristic changes indicative of resorption. In some osteoclasts the ruffled border consisted of numerous slender cytoplasmic projections separated by very narrow spaces or channels while in other osteoclasts it was more open. The ruffled border was commonly surrounded by a transitional zone containing numerous thin filaments. The osteoclast usually had its greatest dimension at the level of the ruffled border and the cytoplasm here contained many bodies and vacuoles but a sparse endoplasmic reticulum. Away from the level of the ruffled border the cytoplasmic vacuoles and bodies were fewer while the endoplasmic reticulum was often more pronounced. Parts of the osteoclasts were usually situated close to a vessel. It is suggested that there is a correlation between the development of the ruffled border and the degree of bone resorption and that osteoclasts without a ruffled border are, at least temporarily, inactive with respect to bone resorption. The numerous cytoplasmic bodies, interpreted as lysosomes, are presumed to be important in the resorption process. The closely adjacent positioning of osteoclasts and vessels may facilitate the transport of resorption products to the blood.This research was supported by the Danish Research Council. Grant no. 512–727, 512–819 and 512–1545.I wish to thank Professor Arvid B. Maunsbach for valuable discussions.     

Downregulation of small GTPase Rab7 impairs osteoclast polarization and bone resorption

During skeletal growth and remodeling the mineralized bone matrix is resorbed by osteoclasts through the constant secretion of protons and proteases to the bone surface. This relies on the formation of specialized plasma membrane domains, the sealing zone and the ruffled border, and vectorial transportation of intracellular vesicles in bone-resorbing osteoclasts. Here we show that Rab7, a small GTPase that is associated with late endosomes, is highly expressed and is predominantly localized at the ruffled border in bone-resorbing osteoclasts. The decreased expression of Rab7 in cultured osteoclasts by antisense oligodeoxynucleotides disrupted the polarization of the osteoclasts and the targeting of vesicles to the ruffled border. These impairments caused a significant inhibition of bone resorption in vitro. The results indicate that the late endocytotic pathway is involved in the osteoclast polarization and bone resorption and underscore the importance of Rab7 in osteoclast function.     

Limited and selective localization of the lysosomal membrane glycoproteins LGP85 and LGP96 in rat osteoclasts

 Monospecific antibodies against two major glycoproteins of rat lysosomal membranes with apparent molecular masses of 96 and 85 kDa, termed LGP96 and LGP85, respectively, were used as probes to determine the expression and distribution of lysosomal membranes in rat osteoclasts. At the light microscopic level, the preferential immunoreactivity for both proteins was found at high levels at the side facing bone of actively bone-resorbing osteoclasts. Osteoclasts detached from bone surface were devoid of immunoreactivity for each protein. At the electron microscopic level, both proteins were exclusively confined to the apical plasma membrane at the ruffled border of active osteoclasts with well-developed ruffled border membrane. No immunolabeling for both proteins was observed in the basolateral membrane and the clear zone of bone-resorbing osteoclasts. The plasma membrane of preosteoclasts and post- and/or resting osteoclasts showed little or no reactivity against these two antibodies. The results indicate that lysosomal membrane glycoproteins are actively synthesized in active osteoclasts, rapidly transported to the ruffled border area, and contribute to the formation and maintenance of the acidic resorption lacuna of osteoclasts. Accepted: 9 December 1998     

Possible role of direct Rac1-Rab7 interaction in ruffled border formation of osteoclasts

Rab7 has been shown to regulate the late steps of the endocytic pathway. In bone-resorbing osteoclasts, it is involved in formation of the ruffled border, which is a late endosomal-like compartment in the plasma membrane. Here we report a new Rab7-interacting protein, Rac1, another small GTPase protein that binds to the GTP-form of Rab7 as found with a two-hybrid system. We demonstrate further that Rab7 colocalizes with Rac1 at the fusion zone of the ruffled border in bone-resorbing osteoclasts. In other cell types, such as fibroblast-like cells, partial colocalization is perinuclear. Because Rac1 is known to control the actin cytoskeleton through its effectors, the Rab7-Rac1 interaction may mediate late endosomal transport between microtubules and microfilaments enabling endosomal vesicles to switch tracks and may thus also regulate ruffled border formation in osteoclasts.     

Immunocytochemical localization of cathepsin D in the rat osteoclast.

We performed immunocytochemical localization of cathepsin D in osteoclasts of the proximal growth plate of the rat femurs using both the avidin-biotin-peroxidase complex method for cryo-semi-thin (1 micron) sections and the colloidal gold-labeled IgG method for K4M ultra-thin sections. At the light microscopic level, cathepsin D immunoreactivity in the osteoclasts appeared at the vesicles, granules, and/or small vacuoles. They were distributed throughout the cytoplasm of each cell and were relatively numerous close to the bone surface. This antigen could not be detected at the eroded bone surface. As for other cells, immunoreactivity was seen only in the lysosomes of osteoblast-like cells. Immunoreactivity in the osteoclasts was stronger and greater in the density and number than in osteoblast-like cells. At the electron microscopic level, osteoclasts with well-developed ruffled border possessed numerous cathepsin D-containing lysosomes, vacuoles, and coated vesicle-like structures. Cathepsin D-containing lysosomes fused with cathepsin-negative vacuoles and formed large secondary lysosomes. Osteoclasts with poorly developed ruffled border possessed fewer cathepsin D-containing lysosomes than those with well-developed ruffled border. No immunogold particles were seen in vacuole-like channel expansions of the ruffled borders, between the channels of the ruffled borders, or on the eroded bone surface. These findings demonstrate that osteoclasts contain a large amount of cathepsin D. They suggest that cathepsin D is necessary for osteoclastic bone resorption, that it plays an indirect rather than direct role.     

Immunocytochemical localization of cathepsin D in the rat osteoclast

Summary We performed immunocytochemical localization of cathepsin D in osteoclasts of the proximal growth plate of the rat femurs using both the avidin-biotin-peroxidase complex method for cryo-semi-thin (1 m) sections and the colloidal gold-labeled IgG method for K4M ultra-thin sections.At the light microscopic level, cathepsin D immunoreactivity in the osteoclasts appeared at the vesicles, granules, and/or small vacuoles. They were distributed throughout the cytoplasm of each cell and were relatively numerous close to the bone surface. This antigen could not be detected at the eroded bone surface. As for other cells, immunoreactivity was seen only in the lysosomes of osteoblast-like cells. Immunoreactivity in the osteoclasts was stronger and greater in the density and number than in osteoblast-like cells. At the electron microscopic level, osteoclasts with well-developed ruffled border possessed numerous cathepsin D-containing lysosomes, vacuoles, and coated vesicle-like structures. Cathepsin D-containing lysosomes fused with cathepsinnegative vacuoles and formed large secondary lysosomes. Osteoclasts with poorly developed ruffled border possessed fewer cathepsin D-containing lysosomes than those with well-developed ruffled border. No immunogold particles were seen in vacuole-like channel expansions of the ruffled borders, between the channels of the ruffled borders, or on the eroded bone surface.These findings demonstrate that osteoclasts contain a large amount of cathepsin D. They suggest that cathepsin D is necessary for osteoclastic bone resorption, that it plays an indirect rather than direct role.     

Atp6v1c1 is an essential component of the osteoclast proton pump and in F-actin ring formation in osteoclasts

Bone resorption relies on the extracellular acidification function of V-ATPase (vacuolar-type proton-translocating ATPase) proton pump(s) present in the plasma membrane of osteoclasts. The exact configuration of the osteoclast-specific ruffled border V-ATPases remains largely unknown. In the present study, we found that the V-ATPase subunit Atp6v1c1 (C1) is highly expressed in osteoclasts, whereas subunits Atp6v1c2a (C2a) and Atp6v1c2b (C2b) are not. The expression level of C1 is highly induced by RANKL [receptor activator for NF-kappaB (nuclear factor kappaB) ligand] during osteoclast differentiation; C1 interacts with Atp6v0a3 (a3) and is mainly localized on the ruffled border of activated osteoclasts. The results of the present study show for the first time that C1-silencing by lentivirus-mediated RNA interference severely impaired osteoclast acidification activity and bone resorption, whereas cell differentiation did not appear to be affected, which is similar to a3 silencing. The F-actin (filamentous actin) ring formation was severely defected in C1-depleted osteoclasts but not in a3-depleted and a3(-/-) osteoclasts. C1 co-localized with microtubules in the plasma membrane and its vicinity in mature osteoclasts. In addition, C1 co-localized with F-actin in the cytoplasm; however, the co-localization chiefly shifted to the cell periphery of mature osteoclasts. The present study demonstrates that Atp6v1c1 is an essential component of the osteoclast proton pump at the osteoclast ruffled border and that it may regulate F-actin ring formation in osteoclast activation.     

Localization of cathepsins B,D, and L in the rat osteoclast by immuno-light and -electron microscopy

The localization of cathepsins B, D, and L was studied in rat osteoclasts by immuno-light and-electron microscopy using the avidin-biotin-peroxidase complex (ABC) method. In cryosections prepared for light microscopy, immunoreactivity for cathepsin D was found in numerous vesicles and vacuoles but was not detected along the resorption lacunae of osteoclasts. However, immunoreactivity for cathepsins B and L occurred strongly along the lacunae, and only weak intracellular immunoreactivity was observed in the vesicles and peripheral part of the vacuoles near the ruffled border. In control sections that were not incubated with the antibody, no cathepsins were found in the osteoclasts or along the resorption lacunae of osteoclasts. At the electron microscopic level, strong intracellular reactivity of cathepsin D was found in numerous vacuoles and vesicles, while extracellular cathepsin D was only slightly detected at the base of the ruffled border but was not found in the eroded bone matrix. Most osteoclasts showed strong extracellular deposition of cathepsins B and L on the collagen fibrils and bone matrix under the ruffled border. The extracellular deposition was stronger for cathepsin L than for cathepsin B. Furthermore cathepsins B and L immunolabled some pits and part of the ampullar extracellular spaces, appearing as vacuoles in the sections. Conversely, the intracellular reactivity for cathepsins B and L was weak: cathepsin-containing vesicles and vacuoles as primary and secondary lysosomes occurred only sparsely. These findings suggest that cathepsins B and L, unlike cathepsin D, are rapidly released into the extracellular matrix and participate in the degradation of organic bone matrix containing collagen fibrils near the tip of the ruffled border. Cathepsin L may be more effective in the degradation of bone matrix than cathepsin B.     

Specific immunocytochemical localization of cathepsin E at the ruffled border membrane of active osteoclasts

The immunocytochemical localization of cathepsin E, a non-lysosomal aspartic proteinase, was investigated in rat osteoclasts using the monospecific antibody to this protein. At the light-microscopic level, the preferential immunoreactivity for cathepsin E was found at high levels in active osteoclasts in the physiological bone modeling process. Neighboring osteoblastic cells were devoid of its immunoreactivity. At the electron-microscopic level, cathepsin E was exclusively confined to the apical plasma membrane at the ruffled border of active osteoclasts and the eroded bone surface. Cathepsin E was also concentrated in some endocytotic vacuoles of various sizes in the vicinity of the ruffled border membrane, some of which appeared to be secondary lysosomes containing the phagocytosed materials. These results strongly suggest that this enzyme is involved both in the extracellular degradation of the bone organic matrix and in the intracellular breakdown of the ingested substances in osteoclasts.     

Electron microscopic and histochemical studies of the mononuclear osteoclast of the mouse.

Osteoclasts collected from the long bones of mice were cultured on dentin slices. To identify osteoclasts, the tartrate-resistant acid phosphatase (TRACPase) activity of cultured cells was histochemically examined by the azo dye method. The TRACPase-positive cells could be distinguished from other cells by light microscopy. The cells were sectioned by alternating semithin and ultrathin sections to observe their ultrastructure and three-dimensional structure. TRACPase activity was detected both in multi-nucleated osteoclasts and in mononuclear cells. Most of the mononuclear TRACPase-positive cells had features similar to preosteoclasts. A mononuclear TRACPase-positive cell was a ruffled border and clear zone was reconstructed three-dimensionally by NIKON COSMOZONE 2SA. The reconstruction showed that this cell possessed a large clear zone and small ruffled border. Under the ruffled border, no lacuna was apparent; but there was disruption of the dentin surface. The results suggest that this cell was a mononuclear osteoclast and that it might have been in the process of making a new lacuna.     

Studies of the mechanism of spleen cell cure for osteopetrosis in ia rats: appearance of osteoclasts with ruffled borders

After ia (osteopetrotic) rats receive whole body radiation and an injection of spleen cells from a normal littermate, the dense, sclerotic skeleton characteristic of osteopetrosis is rapidly remodeled and becomes normal in appearance radiographically and histologically within three weeks. The mechanism of this skeletal transformation has been explored in cured ia rats by light and electron microscopic examination of osteoclasts. In ia rats less than 25 days of age, osteoclasts viewed by electron microscopy lack a ruffled border - the extensive elaboration of plasma membrane next to the bone surface. Cured ia rats have osteoclasts with ruffled borders indistinguishable from those of normal littermates. In ia rats that receive only 600 rads whole body radiation, osteoclasts are still present three weeks later, but appear abnormal by light microscopy, with dense nuclei and lacking cytoplasmic vacuoles next to the bone surface. Cured ia rats have two types of osteoclasts, one type indistinguishable from osteoclasts of normal littermates by light microscopy, the other resembling osteoclasts of ia rats that received radiation only. These data indicate that the mechanism of the spleen cell cure for osteopetrosis in ia rats is rapid remodeling of the skeleton produced by osteoclasts with ruffled borders. Whether normal spleen cells produce these osteoclasts directly by cell division or indirectly by elaboration of some unknown local factor required for formations of ruffled borders by ia osteoclasts is not known.     

Osteoclast cell-surface changes during the egg-laying cycle in Japanese quail

The medullary bone serves as a source of labile calcium mobilized during calcification of the egg shell in birds. Quantitative histological methods demonstrate that the numbers of medullary bone osteoclasts and nuclei per osteoclast remain unchanged during the egg cycle in the Japanese quail (Coturnix). Therefore, cyclic changes in bone resorption cannot be explained by modulations of osteoclasts from and into other bone cells, a mechanism previously suggested for certain species of birds. Rather, dramatic changes in osteoclast cell-surface features occur during the egg cycle, which might account for cyclic variations in resorptive activity. During egg shell calcification, osteoclasts with ruffled borders are closely apposed to bone surfaces; the cytoplasm is rich in vacuoles that contain mineral crystals and seem to derive from the ruffled border. At the completion of egg shell calcification, the ruffled borders and vacuoles move away from the bone surface, although the osteoclast remains attached to the bone along the filamentous or "clear" zone. Associated with the disappearance of the ruffled borders is the appearance of extensive interdigitated cell processes along the peripheral surface of the osteoclast away from the bone. These unusual structures, which may serve as a reservoir of membrane, largely disappear when ruffled borders and associated structures reappear. Therefore, in these hens, the osteoclasts modulate their cell surface rather than their population during the egg cycle.     

Increased synthesis and specific localization of a major lysosomal membrane sialoglycoprotein (LGP107) at the ruffled border membrane of active osteoclasts

The immunocytochemical localization was investigated of a major lysosomal membrane sialoglycoprotein with a molecular mass of 107 kDa, which was designated as LGP107. The study utilized rat osteoclasts with different bone resorbing activity and osteoclast precursors at various stages of differentiation and maturation together with monospecific antibodies to this protein. Despite its localization primarily in lysosomes and endosomes in the other cell types examined, LGP107 was exclusively confined to the apical plasma membrane at the ruffled border of the active osteoclast, where the osteoclast is in contact with the bone surface. The protein was also concentrated in a number of endocytic vacuoles in the vicinity of the ruffled border membrane. However the labeling was not found in the basolateral membranes of the active osteoclast. The ruffled border membrane detached from the bone surface showed a marked decrease in the extent of the immunolabeling. The post-and/or resting osteoclasts, which were located away from the bone surface, were totally devoid of the membraneous localization of LGP107. No definite immunolabeling was found in the immature preosteoclasts. These results indicate that the protein is largely synthesized in the active osteoclast and rapidly translocated to the ruffled border membrane by vectorial vesicle transport. LGP107 is suggested to contribute to the formation and maintenance of the specialized acidic environment for bone resorption.     

Myeloblastic cell line expresses osteoclastic properties following coculture with marrow stromal adipocytes

Osteoclasts are derived from hemopoietic precursors in the marrow. Their differentiation pathway is still underfined, but an important role was obserned for the marrow micrienvironment in the regulation of osteoclasto genesis. various marrow stromal cell subtypes were used to study their possible role in the formation of osteoclasts from myeloblast (M1) cells. Interactions between M1 cell and the 14F1.1 endothelial-adipocyte stromal cell line were demonstrated in a coculture model. M1 cells attached to the adherent layer of 14F1.1 cells and formed distinct focireminiscente of “cobblestone areas.” Follwing these inteactions, M1 Cells developed specific enzymatic activites and became multinucleated. Both monouclear M1 cells became positive to tartrate-resistant acid phosphatase (TRaP) and ATPase, a feature charactreistic of osteoclasts, and were also responsive to calcitonin. Furthermore, they attached to mineralized bone particles and their membrane changed into a ruffled border at the zone of interaction with the bone matrix. We thus demonstrated that marrow endothelial-adipocytes may play a role in regulating the differentiation of myeloblast into osteoclasts.     

Effects of calcitonin on osteoclasts in vivo

Summary Osteoclasts from the tibial metaphyses of young rats treated with porcine calcitonin were studied by electron microscopy. The animals were sacrificed 1 1/2, 4, 8 or 12 hours after injection of the hormone. In survey sections examined by light microscopy the osteoclasts appeared smaller than in control animals. At the ultrastructural level the osteoclasts showed the following alterations: 1) The typical ruffled border was absent. 2) Acid phosphatase was not present in the extracellular space between cell and bone. 3) The number of large vacuoles was decreased and there was no local accumulation of vacuoles in the cytoplasm. 4) The vacuoles did not contain bone crystals. 5) Vacuoles with cell organelles were increased in number. The majority of these vacuoles were identified as autolysosomes because they contained acid phosphatase and the enclosed cell organelles were partially digested. The above changes were present at all time intervals studied.The findings suggest that calcitonin decreases or inhibits bone resorption by osteoclasts. A decreased function of the osteoclasts may contribute to the hypocalcemic effect of the hormone. The increased number of autolysosomes is evidence of an enhanced autophagocytosis. Possible origins of the autolysosomes in osteoclasts are discussed.This research was supported by grants no. 512–819, 512–1545 and 512–1912 from the Danish Medical Research Council. The present observations were first reported at the annual meeting of the Scandinavian Society for Electron Microscopy in Umeå 1973 (Lucht, in press). I wish to thank Professor Arvid B. Maunsbach for valuable discussions and suggestions.     

Ultrastructural evidence in vitro of osteoclast-induced degradation of calcium phosphate ceramic by simultaneous resorption and phagocytosis mechanisms

Osteoclasts are physiological polykaryons specialized in the resorption of calcified tissue. In the context of the clinical use of calcium-phosphate (CaP) ceramics as bone substitutes, this study used transmission electron microscopy to investigate the in vitro mechanisms of CaP ceramic degradation by osteoclastic cell types. Osteoclasts cultured on CaP ceramic developed typical ultrastructural features of bone osteoclasts, such as a polarized dome shape, a clear zone and a ruffled border. Modification of the shape and density of CaP crystals under the ruffled border indicated an acidic microenvironment. Moreover, osteoclasts were able to degrade ceramic by simultaneous resorption and phagocytosis mechanisms. Phagocytosis did not alter the ability of osteoclasts to resorb CaP ceramic. The phagocytosis mechanism consisted of three steps: crystal phagocytosis, disappearance of the endophagosome envelope membrane and fragmentation of phagocytosed crystals within the cytoplasm. The common mechanism of phagocytosis described here is similar to that observed with the monocyte/macrophage lineage, confirming that osteoclasts are part of the mononuclear phagocyte system. Osteoclasts are thus clearly involved in CaP degradation by means of resorption and phagocytosis.     

CELLULAR BIOLOGY OF BONE RESORPTION

Past knowledge and the recent developments on the formation, activation and mode of action of osteoclasts, with particular reference to the regulation of each individual step, have been reviewed. The following conclusions of consensus have emerged.
1. The resorption of bone is the result of successive steps that can be regulated individually.
2. Osteoclast progenitors are formed in bone marrow. This is followed by their vascular dissemination and the generation of resting preosteoclasts and osteoclasts in bone.
3. The exact pathways of differentiation of the osteoclast progenators to mature osteoclasts are debatable, but there is clear evidence that stromal cells support osteoclast generation.
4. Osteoclasts are activated following contact with mineralized bone. This appears to be controlled by osteoblasts that expose mineral to osteoclasts and/or release a factor that activates these cells.
5. Activated osteoclasts dissolve the bone mineral and digest the organic matter of bone by the action of agents secreted in the segregated microcompartments underlying their ruffled borders. The mineral is solubilized by protons generated from CO, by carbonic anhydrase and secreted by an ATP-driven vacuolar H⁺-K⁺-ATPase located at the ruffled border. The organic matrix of the bone is removed by acid proteinases, particularly cysteine-proteinases that are secreted together with other lysosomal enzymes in the acid environment of the resorption zone.
6. Osteoclastic bone resorption is directly regulated by a polypeptide hormone, calcitonin (CT), and locally, by ionized calcium (Ca²⁺) generated as a result of osteoclastic bone resorption.
7. There is new evidence that osteoclast activity may also be influenced by the endothelial cells via generation of products including PG, NO and endothelin.     

Cytoplasmic terminus of vacuolar type proton pump accessory subunit Ac45 is required for proper interaction with V(0) domain subunits and efficient osteoclastic bone resorption

Solubilization of mineralized bone by osteoclasts is largely dependent on the acidification of the extracellular resorption lacuna driven by the vacuolar (H+)-ATPases (V-ATPases) polarized within the ruffled border membranes. V-ATPases consist of two functionally and structurally distinct domains, V(1) and V(0). The peripheral cytoplasmically oriented V(1) domain drives ATP hydrolysis, which necessitates the translocation of protons across the integral membrane bound V(0) domain. Here, we demonstrate that an accessory subunit, Ac45, interacts with the V(0) domain and contributes to the vacuolar type proton pump-mediated function in osteoclasts. Consistent with its role in intracellular acidification, Ac45 was found to be localized to the ruffled border region of polarized resorbing osteoclasts and enriched in pH-dependent endosomal compartments that polarized to the ruffled border region of actively resorbing osteoclasts. Interestingly, truncation of the 26-amino acid residue cytoplasmic tail of Ac45, which encodes an autonomous internalization signal, was found to impair bone resorption in vitro. Furthermore, biochemical analysis revealed that although both wild type Ac45 and mutant were capable of associating with subunits a3, c, c', and d, deletion of the cytoplasmic tail altered its binding proximity with a3, c', and d. In all, our data suggest that the cytoplasmic terminus of Ac45 contains elements necessary for its proper interaction with V(0) domain and efficient osteoclastic bone resorption.     

Transcytosis of calcium from bone by osteoclast-like cells evidenced by direct visualization of calcium in cells

'Transcytosis' of calcium (Ca) from bone by osteoclasts was identified by using a newly developed method that uses fixed or living osteoclast-like cells previously differentiated in vitro, a Ca-specific cell-membrane-impermeable fluorescent dye, and confocal laser scanning microscopy. This method, called the cell-membrane-impermeable dye method, revealed that in fixed osteoclast-like cells, a large quantity of Ca was confined within vacuoles and transported toward the apical cell membrane in the cells. These Ca-confined vacuoles were co-localized with marker proteins of both ruffled border and lysosome. The vacuoles were disrupted when treated with an inhibitor of ruffled border ATPase. In living osteoclast-like cells, Ca-confined vacuoles were again preferentially located at the central region and near the apical cell membrane. These results suggest actual transcytosis of Ca from bone by osteoclasts, and are the first direct evidence of the significant role of osteoclasts in the entire process of Ca metabolism in bone.