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PubMed1999

Extensive clear zone and defective ruffled border formation in osteoclasts of osteopetrotic (ia/ia) rats: implications for secretory function.

The cellular distribution of osteoclast integrin subunits alpha(v) and beta(3), the tissue distribution, and level of the apparent ligand osteopontin (OPN) as well as of the putative regulatory enzyme tartrate-resistant acid phosphatase (TRAP) were studied along with the intracellular distribution of the activation marker c-src in osteopetrotic ia/ia (incisors-absent) mutant rats and their normal littermates. In ia/ia rats, the osteoclasts are incapable of bone matrix resorption. Ultrastructurally the cells exhibit extended clear zones at the expense of ordinary ruffled borders. A secretory dysfunction in the mutant is strongly suggested by the absence of detectable extracellular TRAP, concomitant with an accumulation of the enzyme in abundant small cytoplasmic vesicles. Moreover, TRAP mRNA, protein content, as well as enzymatic activity were elevated. Furthermore, increased levels of integrin subunits alpha(v) and beta(3) were detected at the clear zone of mutant osteoclasts. OPN mRNA levels were elevated in long bones from mutants. In ia/ia rats, immunolabeling for OPN was homogeneously distributed at the surface facing osteoclasts, while in normal littermates it was concentrated at the clear zone area and barely detectable at ruffled borders. The absence of OPN labeling in the abundant, putative intracellular secretory vesicles in mutant osteoclasts suggests that these cells do not produce OPN. The osteoclasts of ia/ia rats appeared to produce and translocate the c-src protein to the cell membrane. In ia/ia a defect ruffled border-formation is observed along with extensive clear zone formation and decreased secretory function. The lesion may be due to a signaling defect, but in that case the defect seems to be located downstream to or not involving the c-src pathway. Our results illustrate the close relationship between secretory function and ruffled border formation in osteoclasts, a relationship that appears to be necessary for proper resorptive function.

Acid Phosphatase↗

PubMed1984

Resorption of vital or devitalized bone by isolated osteoclasts in vitro. The role of lining cells.

The maintainance of resorptive capability towards vital or devitalized bone in osteoclasts isolated from the medullary bone of laying hens and cultured for five days in vitro has been investigated morphologically with the aid of light and transmission electron microscopy. Devitalized bone particles ranging in size from 50 to 100 microns, added to cultures of osteoclasts, were rapidly surrounded by the osteoclasts which, in transmission electron microscopy, showed ruffled borders and clear zones at the surfaces of contact with bone-features typical of resorptive activity. Alternatively osteoclasts were added onto the endosteal surfaces of vital or devitalized diaphyses of quail femurs after removal of the endosteal and periosteal cell layers. The results indicated that, when the vital or devitalized bone surfaces were devoid of cells, the osteoclasts adhered and resorbed bone (as confirmed by transmission electron microscopy). When vital bone of quail was cultured for 24 h before the addition of osteoclasts a new cell layer was formed; it enveloped all bone surfaces and precluded the access of osteoclasts to bone. The role of these lining cells, ultrastructurally indistinguishable from resting osteoblasts, is discussed.

Animals↗

PubMed1989

Different tartrate sensitivity and pH optimum for two isoenzymes of acid phosphatase in osteoclasts. An electron-microscopic enzyme-cytochemical study.

By differentiation of substrate specificity, pH optimum range, and sensitivity to various inhibitors, 2 isoenzymes of acid phosphatase in bone cells have been studied at the electron-microscopic level. When p-nitrophenyl phosphate was used for the substrate, the demonstrable enzyme activity was affected by neither tartrate nor sodium fluoride. The reaction product, when incubated at pH 5-6, was detected in all sites along the pathway for the biosynthesis of acid phosphatase in the osteoclast, including the perinuclear space, cisternae of the endoplasmic reticulum, Golgi complex, various vesicles, and vacuoles. In the osteoclasts attached to bone, the enzymatic activity was demonstrated at the extracellular ruffled border and on the eroded bone surface. Reaction products became confined to lysosomes and extracellular ruffled border when incubated at pH 6-7. Unattached osteoclasts showed a similar intracytoplasmic localization of enzyme as the attached ones, except for the absence of the extracellular enzyme activity. The mononuclear, immature type of osteoclast also resembled the mature osteoclast in terms of enzymatic localization. Except for the osteoclasts, the acid p-nitrophenyl phosphatase activity was restricted to lysosomal vesicles in various bone cells, monocytes, and macrophages. Such activity was inhibited by adding 50 mM tartrate to the p-nitrophenyl phosphate medium. When beta-glycerophosphate or p-nitrocatechol sulfate was the substrate, most of the reaction product was localized intracellularly. Unlike the acid p-nitrophenyl phosphatase, the acid beta-glycerophosphatase or arylsulfatase activity in osteoclasts and other bone cells was inhibited completely by 10 mM tartrate or 10 mM sodium fluoride.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗

PubMed1992

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.

Animals↗

PubMed1994

The effects of bisphosphonates on the resorption cycle of isolated osteoclasts.

Binding sites for wheat germ agglutinin (WGA)-lectin have been shown to become revealed in the demineralized resorption lacunae that osteoclasts excavate on bone substrate. Peroxidase-conjugated WGA-lectin, which binds to bone matrix glycoconjugates and proteoglycans, was used in pit formation assays to assess the activity of isolated osteoclasts cultured on either 3-amino-1,1-hydroxypropylidene-bisphosphonate (APD)-or dichloromethylene bisphosphonate (Cl2MBP)-covered bone slices. Immunofluorescence and histochemical techniques were also used to study the effects of bone-bound bisphosphonates on isolated rat osteoclasts. Neither APD nor Cl2MBP interfered with the special organization of actin or vinculin in osteoclasts when the cells were initializing their resorption cycle. After 24 hours of culture, the number of resorbing osteoclasts increased strongly on control slices, but remained low on either APD- or Cl2MBP-treated slices. At this time, the actin and vinculin rings in osteoclasts also started to exhibit abnormal, more diffuse staining. Both bisphosphonates studied resulted in signs of cytotoxicity: the number of osteoclasts decreased on APD- or Cl2MBP-covered bone during the course of the study and those remaining attached exhibited severe cytoplasmic retractions. The total areas of resorption remained at significantly lower levels in both experimental groups studied, and this was due to decreases in both the number and sizes of individual resorption pits. The size of the most extensive lacunae detected on the Cl2MBP slices did not exceed 5 x 10(3) microns 2, whereas on the control slices, resorption pits bigger than 15 x 10(3) microns 2 were frequently discovered.

Actins↗

PubMed1995

Volumes of chick and rat osteoclasts cultured on glass.

We have examined the relationship between the number of nuclei of an osteoclast and its volume. Chick and rat cells were released from long bones by chopping the shafts and flushing the fragments in Eagle's Minimum Essential Medium with added 10% fetal calf serum. The bone cell suspension was seeded onto glass coverslips. In Experiment 1, rat and chick cells were allowed to settle for 15 minutes, more medium was then added, and the cells were cultured in 5% CO2 at 37 degrees C for 4 hours. In Experiment 2, only rat cells were used, and the cells were cultured in the presence or absence of 10(-6) M 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (APD) in the medium for 4 or 6 hours. The coverslips were washed in 37 degrees C phosphate-buffered saline and fixed for 24 hours in 2.5% glutaraldehyde in isotonic cacodylate buffer (initially 37 degrees C). The chick cells were critical point dried (CPD) or freeze dried (FD); all rat cells were FD. After drying, cells were coated with gold by vacuum evaporation. The volumes and areas of osteoclasts were measured using a video-rate, line-confocal reflection laser scanning microscope and the number of nuclei in each cell was counted. The volumes and volumes per nucleus of the FD cells were larger than those of the CPD cells but there was no significant difference in plan-areas. Rat osteoclasts were larger than chick cells in all the measured parameters except the mean number of nuclei/cell. The correlation coefficients for the areas, volumes, and the numbers of nuclei for rat and chick cells were all high (r > 0.725). The volumes and volumes per nucleus, but not the areas or areas per nucleus, of the osteoclasts cultured with APD were significantly smaller than control cells. We conclude that FD causes less shrinkage than CPD; chick osteoclasts are about two-thirds the size of rat osteoclasts; and 10(-6) M APD caused a reduction of rat osteoclast volume and volume per nucleus of 21%.

Animals↗

PubMed1992

Avian osteoblast conditioned media stimulate bone resorption by targeting multinucleating osteoclast precursors.

Osteoblasts are thought to secrete factors that regulate the rate of osteoclastic bone resorption. We studied the effect of osteoblast conditioned medium on bone degradation by multinucleated osteoclast-like cells generated in vitro from mononuclear precursors and found that the medium stimulates bone degradation primarily through interactions with osteoclast precursors. The conditioned medium also stimulates expression of the osteoclast-specific antigen 121F. The increased bone degradation, but not increased 121F expression, is due to the conditioned medium maintaining activity of the osteoclast precursors. Although the osteoclast precursors exhibit the DNA fragmentation characteristic of apoptosis, the osteoblast conditioned medium does not prevent such fragmentation. Chicken macrophage growth factor neither mimics nor augments the ability of the conditioned medium to stimulate bone degradation. Studies of osteoclast generation or function should carefully consider whether the effects are dependent on the viability of the resorbing cells.

Alkaline Phosphatase↗

PubMed1993

Purified Pasteurella multocida protein toxin reduces acid phosphatase-positive osteoclasts in the ventral nasal concha of gnotobiotic pigs.

To study the in vivo response of conchal (turbinate) osteoclasts to Pasteurella multocida toxin, four gnotobiotic pigs (7 days of age) were inoculated subcutaneously with 0.2 microgram/kg of purified toxin. One toxin-treated pig along with one control pig were necropsied at 2, 5, 9, and 14 days postinoculation. The entire length of nasal concha from the nasal planum toi ethmoid region was removed, blocked by transverse cuts into five areas, decalcified, sectioned, and then stained with tartrate-resistant acid phosphatase (TRAP) to identify osteoclasts. In each section, total area of concha, total osteoclast cytoplasmic area, and number of osteoclasts were determined using an image analysis morphometric unit. Also collected from pigs were blood and serum for complete blood counts, electrolyte levels, liver enzymes, and TRAP levels. Conchal atrophy increased in severity with time after 2 days postinoculation. In general, the ventral conchae from toxin-treated pigs at 9 and 14 days postinoculation had decreased surface area, osteoclast cytoplasmic area, and numbers of osteoclasts. Serum levels of TRAP were mildly elevated when compared with age-matched controls. No other significant alterations in blood cells or chemistries occurred and no lesions were present histologically in tissues (liver, kidney, lung, heart, and spleen) other than concha. This study shows that the P. multocida toxin induces rapid bone resorption and increases serum levels of acid phosphatase but leads to diminished acid phosphatase expression and presumably, numbers of osteoclasts.

Acid Phosphatase↗

PubMed1993

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.

Animals↗

PubMed1993

Osteoclast recruitment in mice is stimulated by (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate.

Though some evidence suggests that bisphosphonates (BPs) act directly on osteoclasts to inhibit bone resorption, other evidence suggests that they inhibit the development of the osteoclast. We found an increase in osteoclast recruitment in 2-day-old mice given (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD). A threefold increase in 5-bromo-2'-deoxyuridine (BrdU)-labeled osteoclast nuclei was observed on mouse parietal bones 3 days after APD injection. This suggests that inhibition of osteoclast development is not an action of APD in mice of this age. The mechanism of the increased recruitment was investigated. As osteoclast progenitors were not detected on parietal bones in vitro, we looked for an increase in circulating monocytes to account for the recruitment. No such increase was found, but when 51Cr-labeled bone marrow was injected intraperitoneally into mice given APD there was an increase in accumulation of 51Cr in calvaria and in femur and tibia over controls. This increase did not occur when 51Cr-labeled erythrocytes or free 51Cr was injected. We conclude that APD causes increased recruitment of osteoclast precursors by increasing the avidity of bone for hematopoietically derived cells.

Animals↗

PubMed1991

Histochemistry of tartrate-resistant acid phosphatase and carbonic anhydrase isoenzyme II in osteoclast-like giant cells in bone tumours.

Using routinely processed, paraffin-embedded tissue specimens, osteoclast-like giant cells in giant cell tumour of bone (GCT), chondroblastoma, osteoblastoma and osteoblastic osteosarcoma were examined histochemically for osteoclast-specific enzymes tartrate-resistant acid phosphatase (TRAP) and carbonic anhydrase isoenzyme II (CA-II). Osteoclast-like giant cells and some mononuclear cells possessed TRAP activity. These were further classified with respect to CA-II immunoreactivity, i.e. cells with CA-II were seen in GCT and chondroblastoma, while those in osteoblastoma and osteoblastic osteosarcoma were negative for CA-II. All the cellular components in malignant fibrous histiocytoma and various extraosseous inflammatory lesions including malignant giant cells and macrophage polykaryons were negative for both TRAP and CA-II. These results indicate that osteoclast-like giant cells in GCT, chondroblastoma, osteoblastoma and osteoblastic osteosarcoma are all osteoclasts and generated by fusion of mononuclear cells with the same histochemical characteristics as osteoclast-like giant cells. The difference in CA-II immunoreactivity suggests the functional or maturational difference between osteoclast-like giant cells in GCT and chondroblastoma and those in osteoblastoma and osteosarcoma.

Acid Phosphatase↗

PubMed1993

Comparative effect of cadmium on osteoblastic cells and osteoclastic cells.

Cadmium(Cd) has been thought to disturb the bone metabolism directly. The mechanism for the bone lesion is unknown, however. To examine the effects of cadmium on bone metabolism, we compared its effects on osteoblasts and osteoclasts in vitro. We used an established cell line, MC3T3-E1, as osteoblasts and tartrate resistant acid phosphatase (TRACP)-positive multi-nucleated cells (MNC) formed by a bone marrow culture system as osteoclasts. Alkaline phosphatase (ALP) activity was decreased by 10(-7) M Cd and DNA content and hydroxyproline content of osteoblastic cells were decreased by 10(-5) M Cd. Cadmium at 10(-7) M inhibited the osteoclastic cell formation from mouse bone marrow in the presence of 10(-8) M 1 alpha, 25(OH)2 vitamin D3. A 100-fold higher concentration of zinc(Zn) simultaneously added to the cadmium-containing medium prevented the toxicity of cadmium to osteoclastic cells as observed in the culture of osteoblastic cells. These results indicate that both bone formation and bone resorption are inhibited by cadmium. The responses of osteoclasts and osteoblasts to cadmium in this culture system were the same and the responses of cadmium-damaged osteoblasts and osteoclasts to zinc were also similar. These results suggest that another mechanism by which cadmium could cause bone damage should be considered in addition to the specific induction of osteoclastic cells by Cd.

Acid Phosphatase↗

PubMed1977

Isolation of osteoclasts by velocity sedimentation at unit gravity.

A method is presented for separating osteoclasts from the heterogeneous population of bone and marrow cells. Cell suspensions were prepared from femora of young rabbits by mechanical dispersion. The starting cell suspension typically contained only 1.0% +/- 0.5 osteoclasts. Following an initial 45 min of unit gravity sedimentation in a lucite chamber osteoclasts were primarily distributed in fractions 2-5. A second 45-min sedimentation of these pooled fractions yielded cell suspensions containing greater than 30% osteoclasts (as much as a 50-fold increase over starting percentages). Linear scan analysis, however, revealed that osteoclasts accounted for 73.14% +/- 0.58 of the cell colume. Subsequent in vitro experiments demonstrated linear incorporation of 3H-leucine into TCA precipitable protein for cells comprising the osteoclast fraction. Concomitant radioautographs revealed radioactive label in isolated osteoclasts.

Animals↗

PubMed1977

Quantitative analyses of osteoclast changes in resorbing bone organ cultures.

The stimulation of bone resorption, assessed by the release of 45Ca from prelabeled bones, was associated with an increase in number of osteoclasts per bone section in parathyroid hormone (PTH)-treated bones, but not in lipopolysaccharide (LPS)-treated bones. By contrast the number of nuclei per osteoclast increased following LPS treatment, but was not affected by PTH. LPS-treated bones had more multinucleated cells, some having as many as 27 nuclei per osteoclast. More osteoclasts were adjacent to the bone collar in bones treated with LPS or PTH than in control bones. In LPS-treated bones this area also contained the largest osteoclasts, as determined by the greatest number of nuclei per osteoclast. The results suggest that LPS and PTH stimulate osteoclastic resorption by different mechanisms.

Animals↗

PubMed1978

Mechanism of osteoclastic bone resorption: a new hypothesis.

Osteoclastic bone resorption involves the solubilization of the mineral salts and the degradation of noncollagen bone matrix and collagen fibrils. As no recognizable collagen fibrils have ever been reported within cytoplasmic vacuoles in osteoclasts, it is generally assumed that the collagen fibrils are digested extracellularly in the resorption zone. The extent to which lysis occurs extracellularly and whether or not the osteoclasts phagocytose the degradation products remain to be established. In the present communication, a hypothesis is presented suggesting the possibility that osteoclastic resorption of bone involves the participation of two different cell types. According to this hypothesis, osteoclastic bone resorption is initiated by osteoclasts that demineralize areas of bone and degrade noncollagen bone matrix. After the osteoclasts have moved away or become partially detached from the demineralized site, the exposed collagen fibrils are phagocytosed by mononuclear, fibroblast-like or monocyte-derived cells.

Bone Matrix↗

PubMed1996

Gold salts inhibit osteoclastic bone resorption in vitro.

Loss of bone mass is commonly associated with rheumatoid arthritis (RA) and is increasingly considered to be due to the increased activity of bone-resorbing osteoclasts. Gold salts such as auranofin (AF), aurothioglucose (ATG) and aurothiomalate (ATM) have beneficial therapeutic effects in RA, but their mechanisms(s) of action is not well understood. In the present study we have examined the effects of these 3 gold salts on osteoclastic bone resorption in vitro, using the bone slice assay where bovine cortical bone slices are resorbed by osteoclasts disaggregated from the long bones of neonatal rats. All 3 gold salts inhibited osteoclastic bone resorption with IC50 values of AF = 0.1 microgram/ml, ATG and ATM = 1 microgram/ml. All 3 compounds caused a decreased survival of osteoclasts on bone slices at high concentrations indicating a cytotoxic effect that was also observed in a cytotoxicity assay with osteoblast-like UMR-106 cells. Preincubation of bone slices with various concentrations of AF followed by extensive washing prior to use in the bone slice assay also resulted in an inhibition of bone resorption (IC50 = 4 micrograms/ml) and osteoclast survival on the bone slices preincubated with high concentrations of AF was decreased. Since these effects were obtained with therapeutic concentrations of gold salts, these results indicate that inhibition of osteoclastic bone resorption by gold salts may, at least in part, account for their beneficial effects in RA.

Animals↗

PubMed1983

Effect of hydrocortisone on osteoclasts generated in cat bone marrow cultures.

The generation of osteoclasts in cultures of cat bone marrow was completely inhibited for 4 weeks with 10(-6)M hydrocortisone (HC) and partially inhibited with 10(-7) to 10(-9)M in a dose-dependent fashion. This effect was completely reversible when cultures were exposed for only 2 weeks to 10(-9) or 10(-8)M HC. However, cultures in which higher concentrations (10(-7) to 10(-5)M) were maintained for the same period did not show complete recovery in terms of numbers of osteoclasts and number of nuclei per cell after withdrawal of HC, suggesting that precursor cells of osteoclasts were also damaged by HC. To study the effects of HC on osteoclasts already present in the cultures, 10(-6)M was added to 4-week-old untreated cultures. The number of osteoclasts decreased rapidly and a gross morphological response was also apparent (rounding of the cells leading to detachment from the substratum and inhibition of cell fusion), indicating that the generation as well as the survival of osteoclasts in vitro are sensitive to HC. The morphological changes observed under optical and electron microscopy correspond to those of the reported inactive form of osteoclasts, and suggest that their function may also be altered by HC.

Animals↗

PubMed1983

Pharmacological control of osteoclastic motility.

We separated osteoclasts from bone and observed the effect of several known and potential mediators of the control of bone resorption on their cytoplasmic motility. We already found that calcitonin (CT), a hormone that inhibits bone resorption, regularly causes complete inhibition of cytoplasmic motility, specific for osteoclasts, through a trypsin-sensitive membrane receptor [1]. We report here that prostaglandin I2 (PGI2) and dibutyryl cyclic AMP induce an identical change in osteoclastic behavior. We found that theophylline, which inhibits intracellular cyclic AMP degradation, and which itself had no effect on osteoclastic motility, potentiated the cytoplasmic inhibition caused by CT, PGI2, and cyclic AMP. This suggests that PGI2 and CT cause cytoplasmic quiescence by increasing the intracellular level of cyclic AMP, a view compatible with the known ability of CT to increase cyclic AMP in bone [2]. Parathyroid hormone (PTH), PGE2, and 1,25 dihydroxycholecalciferol (1,25 (OH)2D3), hormones known to stimulate osteoclasts, did not stimulate the activity of either active or quiescent isolated osteoclasts. The undoubted ability of these hormones to stimulate osteoclastic activity in vivo may therefore be mediated through a primary hormonal interaction with another cell type.

Animals↗