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PubMed2000

Regulation of the differentiation and function of osteoclasts.

The osteoclast is the cell that resorbs bone. It has been known for many years that its formation and function are regulated by cells of the osteoblastic lineage. Recently the molecular basis for this regulation was identified; osteoblastic cells induce osteoclastic differentiation and resorptive activity through expression of tumour necrosis factor (TNF) activation-induced cytokine (TRANCE) (also known as RANKL, ODF, OPGL, and TNFSF11), a novel membrane-inserted member of the TNF superfamily. Osteoclastic regulation is assisted through secretion of an inhibitor, osteoprotegerin (OPG) (OCIF, TNFRSF11B), a soluble (decoy) receptor for TRANCE. Osteoclast formation and survival also depend on and are substantially enhanced by transforming growth factor-beta (TGF-beta), which is abundant in bone matrix. Surprisingly, not only TRANCE but also TNF-alpha can induce osteoclast formation in vitro from bone marrow-derived mononuclear phagocytes, especially in the presence of TGF-beta. Whether or not TNF-alpha does the same in vivo, its ability to generate osteoclasts in vitro has significant implications regarding the nature of osteoclasts and their relationship to other mononuclear phagocytes, and a possible wider role for TRANCE in macrophage pathobiology. A hypothesis is presented in which the osteoclast is a mononuclear phagocyte directed towards a debriding function by TGF-beta, activated for this function by TRANCE, and induced to become specifically osteoclastic by the characteristics of the substrate or signals from bone cells that betoken such characteristics.

Bone Resorption↗
PubMed2000

Synovial tissue in rheumatoid arthritis is a source of osteoclast differentiation factor.

OBJECTIVE: Osteoclast differentiation factor (ODF; also known as osteoprotegerin ligand, receptor activator of nuclear factor kappaB ligand, and tumor necrosis factor-related activation-induced cytokine) is a recently described cytokine known to be critical in inducing the differentiation of cells of the monocyte/macrophage lineage into osteoclasts. The role of osteoclasts in bone erosion in rheumatoid arthritis (RA) has been demonstrated, but the exact mechanisms involved in the formation and activation of osteoclasts in RA are not known. These studies address the potential role of ODF and the bone and marrow microenvironment in the pathogenesis of osteoclast-mediated bone erosion in RA. METHODS: Tissue sections from the bone-pannus interface at sites of bone erosion were examined for the presence of osteoclast precursors by the colocalization of messenger RNA (mRNA) for tartrate-resistant acid phosphatase (TRAP) and cathepsin K in mononuclear cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to identify mRNA for ODF in synovial tissues, adherent synovial fibroblasts, and activated T lymphocytes derived from patients with RA. RESULTS: Multinucleated cells expressing both TRAP and cathepsin K mRNA were identified in bone resorption lacunae in areas of pannus invasion into bone in RA patients. In addition, mononuclear cells expressing both TRAP and cathepsin K mRNA (preosteoclasts) were identified in bone marrow in and adjacent to areas of pannus invasion in RA erosions. ODF mRNA was detected by RT-PCR in whole synovial tissues from patients with RA but not in normal synovial tissues. In addition, ODF mRNA was detected in cultured adherent synovial fibroblasts and in activated T lymphocytes derived from RA synovial tissue, which were expanded by exposure to anti-CD3. CONCLUSION: TRAP-positive, cathepsin K-positive osteoclast precursor cells are identified in areas of pannus invasion into bone in RA. ODF is expressed by both synovial fibroblasts and by activated T lymphocytes derived from synovial tissues from patients with RA. These synovial cells may contribute directly to the expansion of osteoclast precursors and to the formation and activation of osteoclasts at sites of bone erosion in RA.

Arthritis, Rheumatoid↗
PubMed1981

Osteoclast cell-surface specializations and nuclear kinetics during egg-laying in Japanese quail.

Medullary bone deposits serve as a reservoir of labile calcium for egg-shell calcification in birds. Quantitative transmission-electron-microscope methods and light-microscope autoradiographic cell-population-kinetic analyses were used to determine changes in cell-surface specializations and population dynamics of medullary bone osteoclasts during egg-laying in Japanese quail. Prior to egg-shell formation, from 0 to about 8 hours after the previous oviposition, very few osteoclast profiles had ruffled borders. The appearance of ruffled borders coincided with the beginning of egg-shell calcification, about 9-10 hours after the previous oviposition. During egg-shell calcification, about 10-21 hours after the previous oviposition, most osteoclast profiles had ruffled borders. Ruffled borders disappeared at the completion of egg-shell calcification and commencement of egg-shell pigmentation. Thus, functional activities of medullary bone osteoclasts appear to be closely synchronized with egg-shell calcification during egg-laying. From 1 to 48 hours after a single injection of 3H-thymidine (3H-TdR), very few labeled osteoclast nuclei were seen during egg-laying. Following multiple injections of 3H-TdR, the percentage of labeled nuclei reached a peak at about 170 hours after the first injection. At this peak-labeling time, relatively few of the osteoclast profiles that had labeled nuclei had two or more; although the average number of nuclei per osteoclast profile was about 3.6. These kinetic data suggest that the medullary bone osteoclast population has a prolonged rate of turnover compared to rapid changes in cell activities associated with each 24-hour egg-laying cycle; and collectively they would suggest that rapid changes in osteoclast functions occur independently of changes in cell-population dynamics.

Animals↗
PubMed2006

Role of the osteoclast in cranial suture waveform patterning.

This study investigates the role of bone resorption in defining interdigitations characteristic of cranial suture waveform. Male mice from the CD-1 (ICR) background were analyzed at six age groups (n = 5 mice per group) in order to study the ontogenetic changes of osteoclast counts using tartrate-resistant acid phosphatase-stained histological sections of sagittal sutures. Additionally, the complexity of suture lines were measured ectocranially from the same age groups (n = 5 per group) using image capture and fractal geometry (ruler dimension method). The results suggest that osteoclast resorption is a contributor to suture patterning. Specifically, osteoclasts show the greatest activity along concave suture regions at 42 and 84 days (Kruskal-Wallis test statistic = 14.9; P < or = 0.01). This coincides with significant increases incrementally in suture complexity as measured with fractal dimension at 42 and 84 days of age (ANOVA F-statistic = 19.84; P < or = 0.001). In congruence with these data, mice given osteoclast-depleting injections of alendronate show a decrease in sagittal suture complexity. Data from this experiment indicate a positive relationship between suture complexity and osteoclast count (P < 0.01; r = 76%). Increases in suture complexity and osteoclast activity occur after peak rates of cranial width growth and coincide with weaning and the transition to a hard chow diet. These data demonstrate osteoclasts along the bone margin of the cranial suture and also indicate that sutures attain their complex shape at the same age when osteoclast number is highest along concave suture margins, underscoring the role of osteoclasts in generating the suture waveform pattern.

Acid Phosphatase↗
PubMed1990

Myosin II antibodies inhibit the resorption activity of isolated rat osteoclasts.

We present microinjection data in support of an indirect approach by which cytoplasmic protein interactions important in the processes of bone resorption can be elucidated. Three polyclonal antibodies (M1, M3, M5) raised against myosin II from perfused rat liver differently affected the actin-activated Mg ATPase of myosin II. These antibodies microinjected into isolated rat osteoclasts affected osteoclast morphology and activity in bone resorption. M1, which completely inhibited myosin ATPase activity at a antibody:myosin ratio of 10:1, initially promoted the extension/retraction motility of lamellipodia but eventually reduced the spread area of osteoclasts along the substrate after 20 hr. M3, which inhibited ATPase activity by 70%, had similar effects; however, M5, which weakly inhibited ATPase activity, neither promoted extension/retraction nor reduced spread area of osteoclasts. Immunofluorescence showed that these antibodies removed myosin II from the majority of actin filaments in injected osteoclasts. Because antibodies that did not bind to a myosin II column had little effect on the extension/retraction of lamellipodia or the osteoclast spread area, these data suggest that myosin II participates in the stabilization of osteoclast lamellipodia along the substrate. M1 injection strongly inhibited injected osteoclasts from excavating resorption lacunae in bone slices, compared to control antibody. M3 and M5 were less effective but also inhibited bone resorption. These data show that myosin II is functionally important in bone resorption and that the osteoclast-differentiated activity of bone resorption is a more sensitive assay for myosin activity than lamellipodia motility or cell morphology.

Animals↗
PubMed1989

Bone matrix stimulates osteoclastic differentiation in cultures of rabbit bone marrow cells.

Cells showing osteoclastic characteristics have not been identified outside bone. Because osteoclasts originate from an extraosseous source, this suggests that identifiable osteoclastic features do not develop until the precursors enter bone, where the local microenvironment may signal osteoclastic differentiation or maturation. We assessed the influence of bone matrix on osteoclastic differentiation by incubating bone marrow cells, after removal of pre-existing osteoclasts, on plastic coverslips or slices of devitalized cortical bone. We found that there was a threefold increase in the number of osteoclast-specific MAb-positive cells on the bone matrix compared with plastic coverslips. The number of MAb-positive cells correlated with the extent of excavation of the surface of the bone slices. Multinuclearity correlated with MAb-positive cell density, and for any given density the proportion of MAb-positive cells that were multinucleate was similar on plastic and bone. We conclude that, in the presence of 1,25-(OH)2 vitamin D3, bone matrix stimulates the generation of osteoclasts but has no demonstrable influence on the fusion of mononuclear osteoclastic precursors.

Animals↗
PubMed1990

Correlation of an osteoclast antigen and ruffled border on giant cells formed in response to resorbable substrates.

The osteoclast is the specialized multinucleated cell primarily responsible for the degradation of the organic and inorganic components of bone matrix. The functional and developmental relationship between osteoclasts and foreign body giant cells is unclear. The osteoclast plasma membrane ruffled border juxtaposed to the bone surface is a unique morphologic characteristic of active osteoclasts. In the studies reported here giant cell formation was induced in response to a variety of materials implanted onto the richly vascularized chick chorioallantoic membrane. Light and electron microscopic techniques were used to examine the morphologic characteristics of the giant cells. In addition, immunohistochemical methods were used to demonstrate the appearance of a 150 kD cell surface antigen on chicken osteoclasts recognized by monoclonal antibody 121F. Giant cells that formed in response to mineralized bone particles exhibited ruffled borders and stained positively with the 121F antibody. Many giant cells that formed in response to hydroxyapatite possessed ruffled borders similar to but not as extensive as those observed on giant cells formed on bone. Immunohistochemical localization of the 121F antigen on these cells suggested that the antigen was present, but staining intensity was reduced compared to that of bone-associated giant cells. The formation of mineral matrix complexes by the adsorption to hydroxyapatite of bone extract or osteocalcin enhanced ruffled borders and the presence of the 121F antigen on elicited giant cells. In contrast, giant cells that formed on non-resorbable materials, such as Sepharose beads, mica, and methacrylate, lacked ruffled borders and were negative for the 121F antigen. It appears that expression of the 121F osteoclast antigen correlates with the appearance and extent of ruffled membranes on giant cells. Furthermore, it appears that giant cell ruffled membrane development and the presence of the 121F osteoclast antigen are related to giant cell formation in response to resorbable materials that are subject to extracellular dissolution. Expression of this antigen may be indicative of the developmental and/or functional state of giant cells (osteoclasts) that form on resorbable substrates. In addition, components of the bone matrix, including osteocalcin, in association with bone mineral, lead to elevated levels of this osteoclast antigen.

Allantois↗
PubMed1993

Transient recruitment of osteoclasts and expression of their function in osteopetrotic (op/op) mice by a single injection of macrophage colony-stimulating factor.

Severe deficiency of osteoclasts in op/op mice, caused by the absence of functional macrophage colony-stimulating factor (M-CSF), is cured by daily injections of purified recombinant human M-CSF (rhM-CSF). In this study, we found that a single injection of 5 micrograms rhM-CSF is enough for recruitment of osteoclasts in mutant mice. Osteoclast number increased during the period between 2 and 4 days after the single rhM-CSF injection. When YM175, a new derivative of bisphosphonate, was administered to the mice 4 days after rhM-CSF injection or later, osteoclasts disappeared by 3 days after YM175 administration. However, a significant number of osteoclasts were detected even at 3 days after YM175 administration when YM175 was administered 3 days after rhM-CSF injection or earlier. These results indicate that YM175 is cytotoxic only to functioning osteoclasts and that recruitment of osteoclasts is finished 4 days after a single rhM-CSF injection. The osteoclasts actively resorbed bone trabeculae for a prolonged period, demonstrating that M-CSF is not requisite for the functioning of mature osteoclasts.

Animals↗
PubMed1995

Bisphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo.

Bisphosphonates inhibit bone resorption and are therapeutically effective in diseases of increased bone turnover, such as Paget's disease and hypercalcemia of malignancy. The mechanisms by which they act remain unclear. Proposed mechanisms include inhibition of osteoclast formation from precursors and inhibitory or toxic effect on mature osteoclasts. We have developed a new in vitro model to study osteoclast survival and in this paper present in vitro and in vivo evidence that may explain both the observed reduction in osteoclast numbers and in bone resorption by mature osteoclasts, namely that bisphosphonates induce programmed cell death (apoptosis). Three bisphosphonates (risedronate, pamidronate, and clodronate) caused a 4- to 24-fold increase in the proportion of osteoclasts showing the characteristic morphology of apoptosis in vitro. This observation was confirmed in vivo in normal mice, in mice with increased bone resorption, and in nude mice with osteolytic cancer metastases, with similar-fold increases to those observed in vitro. Of the three compounds, risedronate, the most potent inhibitor of bone resorption in vivo, was the strongest inducer of osteoclast apoptosis in vitro. Osteoclast apoptosis may therefore be a major mechanism whereby bisphosphonates reduce osteoclast numbers and activity, and induction of apoptosis could be a therapeutic goal for new antiosteoclast drugs.

Animals↗
PubMed1996

Purification and characterization of fully functional human osteoclast precursors.

The identification and purification of human osteoclast precursors is essential to further our understanding of the mechanisms that control human osteoclast differentiation. Osteoclastoma tissue potentially provides a rich source of human osteoclast precursors, and in previous studies we have demonstrated the existence of a population of mononuclear cells within this tissue that is reactive with osteoclast-selective vitronectin receptor monoclonal antibodies. In this study, mononuclear cells expressing the vitronectin receptor, as defined by their ability to react with a murine monoclonal antibody to the beta 3 chain of the vitronectin receptor (87MEM1), were isolated from collagenase digests of osteoclastoma tissue using a fluorescence activated cell sorter. Based on their fluorescence signal and size, approximately 2-3% of the viable cells (typically 2 x 10(5)) were obtained and prepared for further phenotyping. The isolated cells demonstrated a number of phenotypic characteristics of osteoclasts: positive tartrate-resistant acid phosphatase (TRAP) activity, reactivity with human osteoclast-selective antibodies, expression of calcitonin receptors, cathepsin K (a novel osteoclast-selective cysteine proteinase) mRNA, and osteopontin mRNA and protein. These phenotypic characteristics were also detected in mononuclear cells within cryostat sections of the native osteoclastoma tissue as well as in resorption lacunae of sections of human bone. In contrast, isolated peripheral blood monocytes were negative for TRAP activity and osteopontin expression and, unlike the osteoclastoma-derived cells, demonstrated strong nonspecific esterase activity. Significantly, when the osteoclastoma-derived 87MEM1 positive cells were cocultured on whale dentine for 1-3 weeks with stromal cells, extensive resorption of the dentine surface was observed. This is the first demonstration of the purification of human osteoclast precursors. These cells provide an homogeneous cell population for studying cellular events that occur during human osteoclast differentiation.

Antibodies, Monoclonal↗
PubMed2002

CCL9/MIP-1gamma and its receptor CCR1 are the major chemokine ligand/receptor species expressed by osteoclasts.

Although much has been learned recently of the mechanisms by which the differentiation of osteoclasts is induced, less is known of the factors that regulate their migration and localization, and their interactions with other bone cells. In related cell types, chemokines play a major role in these processes. We therefore systematically tested the expression of RNA for chemokines and their receptors by osteoclasts. Because bone is the natural substrate for osteoclasts and may influence osteoclast behavior, we also tested expression on bone slices. Quantitative RT-PCR using real-time analysis with SYBR Green was therefore performed on RNA isolated from bone marrow cells after incubation with macrophage-colony stimulating factor (M-CSF) with/without receptor-activator of NFkappaB ligand (RANKL), on plastic or bone. We found that RANKL induced expression of CCL9/MIP-1gamma to levels comparable to that of tartrate-resistant acid phosphatase (TRAP), a major specialized product of osteoclasts. CCL22/MDC, CXCL13/BLC/BCA-1, and CCL25/TECK were also induced. The dominant chemokine receptor expressed by osteoclasts was CCR1, followed by CCR3 and CX3CR1. Several receptors expressed on macrophages and associated with inflammatory responses, including CCR2 and CCR5, were down-regulated by RANKL. CCL9, which acts through CCR1, stimulated cytoplasmic motility and polarization in osteoclasts, identical to that previously observed in response to CCL3/MIP-1alpha, which also acts through CCR1 and is chemotactic for osteoclasts. These results identify CCL9 and its receptor CCR1 as the major chemokine and receptor species expressed by osteoclasts, and suggest a crucial role for CCL9 in the regulation of bone resorption.

Animals↗
PubMed2003

High dose M-CSF partially rescues the Dap12-/- osteoclast phenotype.

Osteoclasts are macrophage derived cells and as such are subject to regulation by molecules impacting other members of the immune system. Dap12 is an adaptor protein expressed by NK cells and B and T lymphocytes. Dap12 also mediates maturation of myeloid cells and is expressed by osteoclasts which are dysfunctional in its absence. We find Dap12-/- osteoclast precursors fail to differentiate, in vitro, and the abnormality is partially rescued by high dose M-CSF. The relative paucity of osteoclast number, even in presence of high dose cytokine, is attended by dampened proliferation of precursor cells and their failure to normally migrate towards the osteoclast-recognized matrix protein, osteopontin. Furthermore, Dap12-/- osteoclasts generated in high dose M-CSF fail to normally organize their cytoskeleton. The incapacity of Dap12 null cells to undergo normal osteoclast differentiation is not due to blunted stimulation of major RANK ligand (RANKL) or M-CSF induced signaling pathways. On the other hand, when plated on osteopontin, Dap12-/- pre-osteoclasts do not activate the tyrosine kinase, Syk, which normally binds to the adaptor protein and transmits downstream signals. Attesting to the importance of the Dap12/Syk complex, Syk deficient macrophages do not undergo normal osteoclastogenesis. Furthermore, the same cells plated onto osteopontin, adhere poorly and fail to phosphorylate c-Src or Pyk2, two kinases central to organization of the osteoclast cytoskeleton.

Adaptor Proteins, Signal Transducing↗
PubMed1991

Evidence for an immunological and functional relationship between superoxide dismutase and a high molecular weight osteoclast plasma membrane glycoprotein.

Large multinucleated osteoclasts are the major cells responsible for bone breakdown and have been reported to produce high levels of superoxides which may contribute to the process of bone resorption (Key et al.: J Bone and Mineral Res 4 [suppl. 1]:S206, 1989). Osteoclasts also possess high levels of superoxide dismutase, a protective enzyme capable of converting toxic superoxides to less dtoxic H2O2 (Fridovich: J Biol Chem 264:7761-7764, 1989). The amino acid sequence of manganese and/or iron superoxide dismutase has a conserved region which exhibits substantial homology with a fragment obtained from a high molecular weight osteoclast surface marker glycoprotein which is reactive with monoclonal antibody 121F. In this report, evidence is presented substantiating immunological, biochemical, and functional similarities between the osteoclast membrane antigen recognized by the 121F monoclonal antibody and superoxide dismutase. Western blot and immunoprecipitation studies show that a monospecific polyclonal antibody generated against immunoaffinity purified antigen is cross-reactive with superoxide dismutase. Both the antigen and a high molecular weight superoxide dismutase activity have been detected in osteoclast plasma membrane preparations. The levels of superoxide dismutase activity and the membrane antigen have been found to correlate in antigen depletion studies and in western blots probing osteoclasts and closely related marrow-derived giant cells. Moreover, regions of osteoclast superoxide dismutase activity identified by electrophoretic zymogram analysis have been shown by gel electrophoresis and western blots to contain the high molecular weight antigen, or complexes of the antigen with the 121F monoclonal antibody when these were premixed prior to nondenaturing electrophoresis. It is proposed that the osteoclast plasma membrane possesses a high molecular weight superoxide dismutase activity. Furthermore, it appears that this activity is associated with the osteoclast antigen recognized by the 121F monoclonal antibody.

Animals↗
PubMed1995

Possible involvement of focal adhesion kinase, p125FAK, in osteoclastic bone resorption.

Involvement of tyrosine phosphorylation in osteoclastic bone resorption was examined using osteoclast-like multinucleated cells prepared from co-cultures of mouse osteoblastic cells and bone marrow cells in the presence of 1 alpha,25-dihydroxyvitamin D3. When osteoclast-like cells were plated on culture dishes in the presence of 10% fetal bovine serum, they were sharply stained in their peripheral region by anti-phosphotyrosine antibody. Western blot analysis revealed that 115- to 130-kD proteins were tyrosine-phosphorylated in osteoclast-like cells. Using immunoprecipitation and immunoblotting, one of the proteins with 115-130 kD was identified as focal adhesion kinase (p125FAK), a tyrosine kinase, which is localized in focal adhesions. Immunostaining with anti-p125FAK antibody revealed that p125FAK was mainly localized at the periphery of osteoclast-like cells. Herbimycin A, a tyrosine kinase inhibitor, not only suppressed tyrosine phosphorylation of p125FAK but also changed the intracellular localization of p125FAK and disrupted a ringed structure of F-actin-containing podosomes in osteoclast-like cells. Antisense oligodeoxynucleotides to p125FAK inhibited dentine resorption by osteoclast-like cells, whereas sense oligodeoxynucleotides did not. These results suggest that p125FAK is involved in osteoclastic bone resorption and that tyrosine phosphorylation of p125FAK is critical for regulating osteoclast function.

Animals↗
PubMed1990

Comparison of bone and parathyroid hormone as stimulators of osteoclast development and activity in calvarial cell cultures from normal and osteopetrotic (mi/mi) mice.

Osteoclast development was studied in cell cultures prepared from calvaria of neonatal osteopetrotic (mi/mi) mice or their normal littermates, using tartrate-resistant acid phosphatase (TRAPase), as an osteoclast marker. In cultures from normal mice, treatment with 10 nM PTH for 4-5 days stimulated the formation of osteoclasts. However in cultures from mi/mi mice, this response was only 7% +/- 5% that of normal mice and they were significantly smaller than osteoclasts of normal mice. Mineralized bone particles elicited osteoclast development in cultures from both normal and mi/mi mice, and osteoclast size was identical for both genotypes. Seventy-eight to 96% of the TRAPase-positive cells bound 125I-CT, as demonstrated by autoradiography. 125I-CT binding characteristics were identical in cultures from both genotypes treated with bone particles, exhibiting a Kd of 3.3-3.6 x 10(-10) M. Addition of PTH stimulated 45Ca release from the added bone particles only in the case of cultures prepared from normal mice, and CT inhibited this response. Cells from normal mice were capable of excavating bone from the surface of smooth cortical bone wafers, but such excavations were rarely seen in the case of calvarial cells from mi/mi mice. Thus, PTH-driven differentiation of osteoclasts is arrested in calvarial cell cultures from mi/mi mice, but mi/mi preosteoclasts retain the ability to express certain osteoclast markers in response to bone derived signals. We hypothesize that the lack of activity of mi/mi osteoclasts is due to the failure of mi/mi preosteoclasts to respond appropriately to resorptive agents, or to cytokines elicited by these agents.

Acid Phosphatase↗
PubMed1995

Polymethylmethacrylate-stimulated macrophages increase rat osteoclast precursor recruitment through their effect on osteoblasts in vitro.

An in vitro rat osteoclast precursor model was employed to study the role of macrophages in the osteolysis associated with aseptic loosening of cemented total joint replacements. Bone resorption at the bone-bone cement interface may involve the release of mediators by macrophages in response to phagocytosis of polymethylmethacrylate particles. Two potential pathways for the macrophage-directed bone resorption were studied. An indirect pathway was investigated in which the macrophage response to cement particles was used to stimulate rat osteosarcoma (ROS) 17/2.8 osteoblasts. Osteoblast-soluble factors then were added to osteoclast precursors. In the direct pathway, osteoclast precursors were exposed directly to macrophage-soluble factors released in response to phagocytosis of cement particles. Osteoclast precursors were identified after adherence to polished human dentin slices. Acid phosphatase-positive osteoclasts were counted using light microscopy at x200 magnification. In the indirect pathway, where the macrophage response was mediated through the rat osteosarcoma osteoblasts, a significant increase in the recruitment of osteoclast precursors was observed. In the direct pathway, when the macrophage-conditioned medium was allowed to interact directly with osteoclast precursors, the adherence of the precursors was significantly decreased. This demonstrates that the macrophage mediators released following phagocytosis of polymethylmethacrylate particles affect the release of soluble factors from osteoblasts. In turn, these osteoblast factors stimulate recruitment of osteoclast precursors to calcified tissue. Evidence from this in vitro model reveals that macrophage-soluble factors, in the absence of an osteoblast contribution, decrease the adherence of osteoclast precursors to calcified substrate. We propose that bone resorption at the aseptically loose interface of cemented arthroplasty may be mediated, at least in part, by soluble factors secreted by osteoblasts responding to macrophages that have phagocytosed particles of polymethylmethacrylate cement.

Animals↗
PubMed2005

Proteomic identification of the TRAF6 regulation of vacuolar ATPase for osteoclast function.

Osteoclasts are cells specialized for bone resorption. For osteoclast activation, tumor necrosis factor receptor-associated factor 6 (TRAF6) plays a pivotal role. To find new molecules that bind TRAF6 and have a function in osteoclast activation, we employed a proteomic approach. TRAF6-binding proteins were purified from osteoclast cell lysates by affinity chromatography and their identity was disclosed by MS. The identified proteins included several heat shock proteins, actin and actin-binding proteins, and vacuolar ATPase (V-ATPase). V-ATPase, documented for a great increase in expression during osteoclast differentiation, is an important enzyme for osteoclast function; it transports proton to resorption lacunae for hydroxyapatite dissolution. The binding of V-ATPase with TRAF6 was confirmed both in vitro by GST pull-down assays and in osteoclasts by co-immunoprecipitation and confocal microscopy experiments. In addition, the V-ATPase activity associated with TRAF6 increased in osteoclasts stimulated with receptor activator of nuclear factor kappaB ligand (RANKL). Furthermore, a dominant-negative form of TRAF6 abrogated the RANKL stimulation of V-ATPase activity. Our study identified V-ATPase as a TRAF6-binding protein using a proteomics strategy and proved a direct link between these two important molecules for osteoclast function.

Actins↗
PubMed1997

A novel culture system to generate osteoclasts and bone resorption using porcine bone marrow cells: role of M-CSF.

A novel osteoclast generation and bone resorption assay system is described in which enhanced osteoclastic generation and bone resorption is induced in porcine bone marrow cell cultures cultured in low-serum medium supplemented with fibroblastic cell (L929) conditioned medium (CM). Numerous osteoclasts, which could be identified by TRAP staining and specific labelling with 121F antibody, were generated in a time-dependent and L929-CM concentration-dependent fashion. A specific antibody against murine M-CSF/CSF-1 abolished osteoclast formation indicating that M-CSF is the essential component of the L929-CM driven osteoclast generation. Culturing on devitalized bone slices resulted in extensive osteoclast-mediated resorption as visualized microscopically. After 16 days in culture, practically the entire bone slice surface was excavated by the osteoclastic cells. Bone resorption could be monitored with time using a novel enzyme-linked immunoassay measuring type I collagen N-telopeptides in culture supernatants. Release of collagen fragments from the slices was paralleled by osteoclastic secretion of TRAP. Salmon calcitonin significantly inhibited collagen fragment and TRAP release. 1,25-Dihydroxyvitamin D3 greatly promoted osteoclast generation and subsequent bone resorption, but its presence was not essential for this process to occur.

Acid Phosphatase↗