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PubMed2006

Secretion of tartrate-resistant acid phosphatase by osteoclasts correlates with resorptive behavior.

There have been dramatic advances recently in our understanding of the regulation of osteoclastic differentiation. However, much less is known of the mechanisms responsible for the induction and modulation of resorptive behavior. We have developed a strategy whereby osteoclasts can be generated in vitro and released into suspension in a fully-functional state. We now exploit this approach to show that tartrate-resistant acid phosphatase (TRAP) is released by osteoclasts during bone resorption. TRAP release was inhibited by the secretion-inhibitor Brefeldin A, and was not accompanied by LDH release. This suggests that TRAP release is due to secretion, rather than cell death. Consistent with this, TRAP secretion was stimulated by resorbogenic cytokines, was inhibited by the resorption-inhibitor calcitonin, and correlated with excavation of the bone surface. We found that, in contrast to incubation on bone, incubation on plastic, glass, or vitronectin-coated plastic substrates did not induce secretion of TRAP. This suggests that the induction of resorptive behavior in osteoclasts depends upon stimulation by bone matrix of a putative osteoclastic "mineral receptor." Release of TRAP by osteoclasts thus represents not only a productive approach to the analysis of the mechanisms that modulate the rate of resorptive activity, but also a system whereby the mechanism through which bone substrates induce resorptive behavior can be identified.

Acid Phosphatase↗

PubMed1995

Antithetic effects of ryanodine and ruthenium red on osteoclast-mediated bone resorption and intracellular calcium concentrations.

In the process of bone remodeling, osteoclasts are responsible for resorption of bone. High levels of intracellular calcium decrease the bone resorbing activity of osteoclasts and increase detachment of osteoclasts from the bone surface. The regulatory role of intracellular calcium in bone resorption is not clearly understood. To understand this phenomenon, we studied the effects of the intracellular calcium modulators ryanodine and ruthenium red on bone resorption using the disaggregated osteoclast pit assay. Changes in intracellular calcium concentrations after treatment with these compounds were detected with the fluoroprobe fura2. With ryanodine, a significant, dose-dependent decrease in bone resorption was detected. This inhibition of bone resorption was reversible upon the removal of ryanodine. Ryanodine increased intracellular calcium concentrations, suggesting that the mechanism of inhibition by ryanodine was via alterations in intracellular stores of calcium. After treatment with ruthenium red, osteoclasts resorbed significantly more bone compared to vehicle-treated cells. This increase in bone resorption correlated with a decrease in intracellular calcium concentrations. The addition of parathyroid hormone or ruthenium red to osteoclast cultures containing ryanodine did not attenuate the decrease in bone resorption caused by ryanodine, suggesting that the mechanism of ryanodine inhibition of bone resorption may involve the "locking" of a calcium channel in an open position.

Analysis of Variance↗

PubMed2003

Hypoxia is a major stimulator of osteoclast formation and bone resorption.

Hypoxia is known to act as a general stimulator of cells derived from marrow precursors. We investigated the effect of oxygen tension on the formation and function of osteoclasts, the cells responsible for bore resorption, which are of promonocytic origin. Using 7- and 13-day cultures of mouse marrow cells on ivory discs, we found that reducing oxygen tension from the ambient atmospheric level of 20% by increasing the proportion of nitrogen caused progressive increases in the formation of multinucleated osteoclasts and resorption pits. Peak effects occurred in 2% oxygen, where stimulations of resorption up to 21-fold were measured. Significant stimulations of osteoclast formation and resorption were observed even in severely hypoxic cultures gassed with 0.2% oxygen. Short-term cultures of cells disaggregated from rat bones indicated that hypoxia did not alter the resorptive activity of mature osteoclasts, but reduced their survival or adherence. In 3-day organ cultures of mouse calvarial bones, exposure to 2% oxygen resulted in maximal, fivefold stimulation of osteoclast-mediated calcium release, an effect equivalent to that of prostaglandin E(2) (PGE(2)), a reference osteolytic agent. Hypoxia also caused a moderate acidosis in calvarial cultures, presumably as a result of increased anaerobic metabolism; this observation is significant because osteoclast activation is dependent on extracellular acidification. Our experiments reveal a previously-overlooked mechanism of considerable potential importance for the regulation of bone destruction. These findings may help explain the bone loss associated with a wide range of pathological states involving local or systemic hypoxia, and emphasize the importance of the vasculature in bone.

Animals↗

PubMed1986

Electrophysiological responses of osteoclasts to hormones.

Electrophysiological measurements were carried out on osteoclasts in vitro. Such isolated osteoclasts are able to resorb bone in vitro and contract in response to calcitonin (CT). Our measurements show that individual osteoclasts respond to CT with a significant transient hyperpolarization of membrane potential. Application of parathyroid hormone (PTH) and dibutyryl cAMP produced a transient hyperpolarization in some osteoclasts. Measurements on an osteoblastlike line (ROS 17/2.8) showed a sustained hyperpolarizing response to CT, which is similar to but smaller than the hyperpolarizing response to PTH and dibutyryl cAMP in this and some other osteoblastlike lines. In contrast to osteoblastlike cells, the osteoclasts have no long term membrane potential response to CT, to PTH, or to dibutyryl cAMP. These results show that there are distinct differences between osteoclasts and osteoblasts in their ion transport responses to hormones.

Animals↗

PubMed1988

Receptor-mediated uptake of a mannose-6-phosphate bearing glycoprotein by isolated chicken osteoclasts.

We have recently shown that degradation of bone collagen by osteoclasts occurs via proteolytic enzyme activity that depends on an acidic milieu. Since bone resorption occurs in an extracellular, acidic compartment located at the cell-matrix attachment site, the osteoclast must deliver the acid collagenolytic enzymes to the cell surface. These observations raise the possibility that the mannose-6-phosphate (M-6-P) receptor, known to sort acidic proteases in other cells, is involved in trafficking lysosomal enzymes to the plasmalemma of bone resorbing cells. To this end we studied receptor-mediated uptake, distribution and release, by isolated chicken osteoclasts, of 125I-hexosaminidase, a M-6-P bearing enzyme. We found that at 4 degrees C, the bone-resorbing polykaryons bind approximately 10,000 molecules of radioligand/cell with a Kd of 0.7 nM, which is endocytosed by osteoclasts at 37 degrees C by a calcium-independent process. Furthermore, 125I-hexosaminidase uptake is unaffected by mannosylated albumin, documenting specificity of the receptor-mediated event. Release of endocytosed enzyme from the cell is also much more rapid than its degradation, attesting to a pathway of uptake and secretion. By autoradiography, the M-6-P bearing ligand is concentrated at the site of osteoclast-bone attachment. Thus, osteoclasts also have the capacity to deliver M-6-P bearing degradative enzymes to their surface at the site of matrix degradation.

Animals↗

PubMed1992

Degradation of collagen in the bone-resorbing compartment underlying the osteoclast involves both cysteine-proteinases and matrix metalloproteinases.

The site of action of cysteine-proteinases (CPs) and matrix metalloproteinases (MMPs) in the degradation of bone collagen by osteoclasts was investigated by evaluating the effects of the CP-inhibitor trans-epoxy-succinyl-L-leucylamido (4-guanidino)-butane (E-64) and the MMP-inhibitor N-(3-N-benzyloxycarbonyl amino-1-R-carboxypropyl)-L-leucyl-O-methyl-L-tyrosine N-methylamide (Cl-1) in an in vitro model system of PTH-stimulated mouse calvaria. In the presence of each of the two inhibitors a large area of collagen free of mineral crystallites was seen adjacent to the ruffled border of the osteoclasts. Following a culture period of 24 h this area proved to be about 10 times larger in inhibitor-treated explants than in controls. Moreover the percentage of osteoclasts in close contact with such demineralized bone areas appeared to be significantly higher in inhibitor-treated explants than in control specimens (60% and 5%, respectively). These effects were not apparent when the osteoclastic activity was inhibited with calcitonin. No significant differences were found between the effects of the two inhibitors, E-64 and Cl-1. Our observations indicate that under the influence of inhibitors of MMPs and CPs demineralization of bone by osteoclasts proceeded up to a certain point whereas matrix degradation was strongly inhibited. It is concluded that within the osteoclastic resorption lacuna both CPs and MMPs participate in the degradation of the collagenous bone matrix.

Animals↗

PubMed2001

Carbonic anhydrase II is an AP-1 target gene in osteoclasts.

c-Fos, a member of the AP-1 family of transcription factors, is necessary for osteoclast differentiation but to date, none of the osteoclast-phenotypic markers have been identified as AP-1 target genes. Here, we demonstrate that carbonic anhydrase II (CA II), an enzyme necessary for osteoclast activity, is transcriptionally upregulated by c-Fos/AP-1. A functional AP-1 binding site is present in the CA II promoter and is necessary for this regulation. Furthermore, we show that AP-1 binding activity, mainly composed of Fra-2 and JunD, is induced by treatment of bone marrow cultures with the osteoclastogenic hormone 1,25 dihydroxyvitamin D(3). Fra-2 and JunD are found in mature osteoclasts as well. Thus, our data demonstrate that cFos/AP-1 can directly regulate the expression of this osteoclast marker and that AP-1 activity is upregulated in osteoclast progenitors in response to osteoclastogenic signals.

Animals↗

PubMed2003

Transcription and immunolocalization of Runx2/Cbfa1/Pebp2alphaA in developing rodent and human craniofacial tissues: further evidence suggesting osteoclasts phagocytose osteocytes.

Runx2/Cbfa1 is a transcription factor, essential for the osteogenic/chondrogenic and odontogenic lineage. Three isoforms of Cbfa1 have been identified, type I (Pebp2alphaA isoform), type II (til-1 isoform), and type III (Osf2 isoform). Here we examined the expression of the Runx2/Cbfa1 during intramembranous and enchondral bone formation in the craniofacial tissues of neonatal rodents (hamster, rat, mouse) and the human fetus. We used a monoclonal antibody raised against the Pebp2alphaA portion and thus potentially recognizing all three isoforms of Runx2/Cbaf1. We report Cbfa1 at the mRNA and protein level in periosteum, preosteoblasts, osteoblasts, young osteocytes, perichondrium, resting and hypertrophic chondrocytes. During active bone remodeling, almost one third of tartrate resistant acid phosphatase (TRAP) positive multinuclear cells identified as osteoclasts were also stained with anti-Pebp2alphaA antibodies. Osteoclasts, however, did not express mRNA transcripts of the Pebp2alphaA gene. Some of the immunopositive structures within these osteoclasts resembled (ingested) cells. TRAP-positive mononuclear cells not attached to bone surfaces did not stain with anti-Pebp2alphaA antibodies. We concluded that the tissue distribution of Runx2/Cbaf1/Pebp2alphaA in ossifying bones of the human fetus is similar to that in neonatal rodent tissues. Osteoclasts do not transcribe the Runx2/Cbfa1 gene but become immunostained by phagocytosing and digesting osteocytes/hypertrophic chondrocytes. The substantial number of osteoclasts involved in phagocytosis of Runx2/Cbfa1 immunopositive cells suggests that phagocytosis is a major way of removing osteocytes/hypertrophic chondrocytes during resorption of bone and cartilage. Finally, the data indicate that positive immunostaining of osteoclasts for typical osteogenic/chondrogenic markers has to be interpreted with caution due to the phagocytosing capacity of these cells.

Animals↗

PubMed1982

Adhesion of osteoclasts and monocytes to developing bone.

Osteoclast resorption of bone matrix during bone development is preceded by cell attachment to bone. An in vitro assay is described that measures adhesion of isolated osteoclasts and their presumed progenitor cell, the monocyte, to embryonic chick bone. Osteoclasts were isolated from day 19 chick tibia and enriched. Circulating chick hatchling monocytes were purified by Ficoll-hypaque sedimentation and attachment to plastic. Isolated cells were labeled with (32P)-KH2PO4 to quantitate cell attachment. In one series of experiments, labeled cells were inoculated into vessels containing day 19 tibias with either endosteal or periosteal bone surface exposed. Labeled cells were also inoculated into chambers containing day 6 (cartilage cores), day 12 (bone and cartilage cores), or day 19 embryonic tibias (bone). Cultures of stage 24 chick limb mesenchyme and embryonic chick skin fibroblasts served as controls. Results demonstrate that twice as many osteoclasts and monocytes adhere to bone as compared to fibroblasts and stage 24 limb mesenchymal cells. Furthermore, there does not appear to be selective adhesion to the endosteal as compared to periosteal bone surfaces. When the data is calculated on the basis of cell attachment per area of each substrate, the day 12 cores had slightly higher number of osteoclasts and monocytes attached compared to day 19 bones; day 6 cartilage cores bound few cells. These observations suggest that osteoclasts and monocytes have a high affinity to bone which seems to be influenced by the developmental age and composition of the substrate.

Animals↗

PubMed1997

Osteoclasts, macrophages, and the molecular mechanisms of bone resorption.

The osteoclast is a physiological polykaryon and the major if not exclusive resorptive cell of bone. It participates in bone remodeling, repair, and growth and mobilization of mineral to meet homeostatic demands. Most importantly, osteoporosis, a disease endemic in Western society and Asia, is always a reflection of enhanced osteoclastic activity relative to bone formation by osteoblasts. In fact, all forms of anti-osteoporosis therapy proven successful involve inhibition of osteoclastic bone resorption. Bone resorption is regulated either by altering recruitment of osteoclast precursors into fully differentiated resorptive polykaryons or modulating the rate at which mature osteoclasts degrade bone. With this in mind, our laboratory has focused on the molecular mechanisms of osteoclast differentiation and the means by which the cell degrades bone matrix.

Animals↗

PubMed1984

Cellular and subcellular events of bone resorption in human and experimental cholesteatoma: the role of osteoclasts.

An ultrastructural study of the interface between aural cholesteatoma and adjacent bone was performed on specimens obtained from human and experimental gerbilline cholesteatoma. When an enlarging cholesteatoma contacted bone, a large number of monocytes and macrophages accumulated in the area of contact. The intervening middle ear mucosa degenerated and bone erosion occurred. Anatomic evidence is provided which indicates that bone erosion in both human and experimental cholesteatoma occurred as a result of the action of multinucleated osteoclasts. These osteoclasts had the ultrastructural appearance of normal osteoclasts with a well developed ruffled border and large numbers of mitochondria. These cells stained readily with acid phosphatase stains. Although many other mononuclear cells were seen in the vicinity of the eroded bone, only the multi-nucleated osteoclasts were associated with disappearance of the bone surface. Osteoclasts were commonly found in experimental cholesteatoma, but were infrequent in human cholesteatoma biopsies, presumable because patients undergoing cholesteatoma surgery ara often treated with topical installations of corticosteroids which inhibit osteoclasts.

Animals↗

PubMed2006

Prostate-specific antigen induces apoptosis of osteoclast precursors: potential role in osteoblastic bone metastases of prostate cancer.

BACKGROUND: Prostate cancer is frequently associated with bone metastases with marked osteoblastic changes and low osteoclastic activity but its mechanism is not well understood. We previously reported that prostate-specific antigen (PSA) stimulated the proliferation and the activation of osteoblasts. In this study, we investigated the effect of PSA on osteoclastogenesis. METHODS: Two human prostate cancer cell lines and PSA were directly injected into human adult bone (HAB) implanted into NOD/SCID mice, followed by morphological analysis. RAW 264.7 cells, murine osteoclast precursor, were treated with PSA. RESULTS: PSA-producing LNCaP and PSA caused a significant decrease of osteoclast precursors and osteoclasts in HAB accompanied by osteoblast proliferation and new bone formation, while PSA-nonproducing PC3 showed increasing osteoclasts with osteolysis. PSA induced apoptosis of RAW 264.7 cells in vitro. PSA-induced apoptosis was dependent of enzymatic activity of PSA and was specific to immature tartrate-resistant acid phosphatase-negative mononuclear RAW 264.7 cells. CONCLUSIONS: PSA plays a crucial role for osteoblastic bone metastasis by promoting both osteoblasts proliferation and apoptosis of osteoclast precursors.

Adenocarcinoma↗

PubMed1997

Conditions that support long-term production of osteoclast progenitors in vitro.

To better understand the mechanisms of osteoclast precursor development from hematopoietic stem cells, we examined the conditions that support the production of osteoclast progenitors, osteoclast colony-forming units (CFU-O), from long-term bone marrow cultures established under myeloid (Dexter's) and lymphoid (Whitlock and Witte's) conditions. Nonadherent cells harvested weekly from myeloid or lymphoid long-term cultures were assayed for CFU-O-derived colony formation in agar in the presence of a murine osteoclast colony-stimulating factor. The myeloid system supported CFU-O production for weeks, but the system produced many other types of myeloid colonies and cells as well, and quantification of CFU-O-derived colonies was difficult. The lymphoid long-term culture system also produced CFU-O; however, CFU-O production in the lymphoid system appeared more selective than in the myeloid system, but was transient. Interestingly, the addition of medium containing G-CSF to these cultures greatly enhanced (> 200%) the CFU-O production. This enhanced CFU-O production was confirmed using bone marrow cultures established on a defined marrow stromal cell line under lymphoid conditions and supplemented with recombinant murine G-CSF. Thus, G-CSF facilitates the development of clonogenic osteoclast progenitors from hematopoietic stem cells in lymphoid long-term culture conditions. This culture system may serve as a useful model for ex vivo generation of osteoclast progenitors.

Animals↗

PubMed1993

Effects of macrophage colony stimulating factor and granulocyte-macrophage colony stimulating factor on osteoclastic differentiation of hematopoietic progenitor cells.

Although the hematopoietic origin of the osteoclast is generally accepted, the precise phenotype of the progenitor and the regulation of its differentiation are unclear. This study compares proliferation and differentiation of progenitors in response to macrophage colony stimulating factor (M-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF). Nonadherent progenitor cells from murine long-term bone marrow cultures (LTBMC) (as a source of osteoclast progenitors) demonstrated a significant proliferative response to M-CSF. In addition, M-CSF increased the number of multinucleated cells, only a small percent of which (14-16%) were tartrate-resistant, acid phosphatase (TRAP)-positive. In contrast, cells cultured with GM-CSF generated more TRAP-positive multinucleated cells even at concentrations less stimulatory of proliferation than M-CSF. The osteoclast phenotype of these multinucleated cells was also assessed by ultrastructural characterization of ruffled borders in association with bone fragments. The bone-active hormone 1,25-dihydroxyvitamin D3 inhibited the proliferation of this subset of progenitor cells in the presence of M-CSF or GM-CSF. All of these results show effects on progenitors in the absence of the stromal cell microenvironment in this system. These results provide evidence for a divergence in the biological responsiveness of osteoclast progenitor cells to M-CSF compared with GM-CSF; they support the notion that M-CSF has a "priming" effect on osteoclast progenitors whose subsequent differentiation to osteoclastic multinucleated cells is promoted by GM-CSF.

Animals↗

PubMed1993

Involvement of dual signal transduction systems in the stimulation of osteoclast-like cell formation by parathyroid hormone and parathyroid hormone-related peptide.

The present study was performed to examine whether parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) would stimulate osteoclast-like cell formation via soluble factor(s) released from osteoblasts and, if so, to characterize the involvement of PTH/PTHrP-responsive dual signal transduction systems [cAMP-dependent protein kinase (PKA) and calcium/protein kinase C(PKC)]. Osteoblasts-conditioned medium (CM) was obtained from rat osteoblastic osteosarcoma cells (UMR-106 cells), which had been cultured in serum free medium for 24 hrs after treatment with various kinds of reagents. The CM of osteoblasts treated with either 10(-7) M human(h) PTH-(1-34) or 10(-7)M hPTHrP-(1-34) equally stimulated osteoclast-like cell formation from hemopoietic blast cells derived from mouse spleen cells, although the CM treated with 10(-8) M 1,25dihydroxyvitamin D3 failed to affect it. The CM treated with both 10(-4) M dibutyryl-cAMP and a direct PKA activator, 10(-4)M Sp-cAMPS significantly increased osteoclast-like cell formation. The CM treated with a PKC activator, 10(-7)M phorbol 12-myristate 13-acetate (PMA) and calcium ionophores (10(-7)M A23187 and 10(-7)M ionomycin) also significantly enhanced osteoclast-like cell formation. The present study first indicated that osteoblast-mediated stimulation of osteoclast-like cell formation by PTH and PTHrP, and the participation of PTH/PTHrP-responsive dual signal transduction systems of osteoblasts in the stimulation of osteoclast-like cell formation by PTH and PTHrP.

Animals↗

PubMed1994

Regulation of osteoclastic acid secretion by cGMP-dependent protein kinase.

Nitric oxide (NO) down-regulates osteoclastic activity. The mechanism is unknown, although, in some cells NO acts by stimulating guanylate cyclase which activates cGMP-dependent proteins. We demonstrated cGMP-dependent protein kinase in osteoclasts by immunofluorescence microscopy. Specificity was confirmed by Western blot analysis showing a single 78 kDa band, the size of the Type I isoform, in isolated avian osteoclasts. Osteoclast function centers on HCl secretion at a specialized membrane organelle. We found that purified cGMP-dependent protein kinase inhibits ATP-dependent acid transport in reconstituted osteoclast membrane vesicles >90%, while cAMP-dependent kinase catalytic subunit, calmodulin kinase II, or cGMP alone were ineffective. This novel, direct modulation of acid transport by cGMP-dependent kinase and the occurrence of the enzyme in osteoclasts suggest that a mechanism of NO-regulation of bone turnover is via cGMP and cGMP-dependent protein kinase inhibition of HCl transport.

Animals↗

PubMed1995

The role of reactive oxygen intermediates in osteoclastic bone resorption.

Osteoclasts have been shown to produce reactive oxygen intermediates (ROI) and it has been suggested that ROI are involved in the process of bone resorption. ROI have also been shown to play a central role in the activation of the multisubunit transcription factor NF-kappa B that enhances the transcription of genes encoding defence and signaling proteins. Therefore, we have assessed the effect of pyrrolidine dithiocarbamate (PDTC), an oxygen-radical scavenger and metal chelator that is a selective and potent inhibitor of NF-kappa B activation, on osteoclastic bone resorption in the bone slice assay. PDTC (0.001-0.1 mM) dose-dependently and non-cytotoxically inhibited osteoclast activity with an IC50 of 0.01 mM. PDTC (0.01 mM) caused no change in the ratio of resorption pit area to resorption pit depth as measured by Lasertec confocal microscopy, indicating that ROI are not involved in the resorptive process per se. This view is supported by time-course studies showing that addition of PDTC or N-acetyl cysteine (NAC; an ROI scavenger, but not metal chelator), 6 hr after the start of the assay had no significant effect on subsequent bone resorption. Desferal (100 microM), a chelator of iron and other metal ions, had no significant effect on bone resorption, indicating (along with the results with NAC) that ROI-scavenging rather than metal chelation is responsible for inhibition of osteoclastic bone resorption by PDTC. Taken together these results indicate that ROI produced by osteoclasts in the bone slice assay are not involved in the process of bone resorption, but are important during osteoclast activation for bone resorption, possibly being involved in activation of the transcription factor NF-kappa B.

Acetylcysteine↗

PubMed1997

Concanavalin A directly stimulates bone-resorbing activity of osteoclasts and their gene expression of cathepsin K/OC-2.

Concanavalin A (Con A), a plant lectin that recognizes cell-surface glycoproteins, up-regulates osteoclastic bone-resorbing activity of cultured isolated rabbit pure osteoclasts as well as unfractionated bone cells. The effect of Con A was blocked by alpha-methyl mannopyranoside. Several Con A-binding proteins were detected in the plasma membranes from osteoclasts. Furthermore, Con A increased the levels of transcripts of cathepsin K/OC-2, one of the proteases responsible for osteoclastic bone-resorbing activity. These results indicate that Con A directly enhances the function of osteoclasts by the association with surface glycoproteins of osteoclasts.

Animals↗