SEARCH · Search PubMed

Results for “OSTEOCLASTS”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 541 records · Page 30Linked to original sources

PubMed2002

Adenosine triphosphate stimulates human osteoclast activity via upregulation of osteoblast-expressed receptor activator of nuclear factor-kappa B ligand.

Nucleotides such as adenosine triphosphate (ATP) and uridine triphosphate (UTP) exist in the extracellular environment where they are agonists at P2 receptors. Both P2Y G-protein-coupled receptors and P2X ligand-gated ion channels are expressed by osteoblasts and osteoclasts, reflected in the diverse nucleotide-induced effects reported to occur in bone. Previous reports have implicated ATP as a proresorptive agent; however, these studies were unable to determine whether ATP mediated its actions directly on osteoclasts, or indirectly via osteoblasts. The development of techniques to generate human osteoclasts in vitro has allowed us to further investigate the intriguing role of extracellular nucleotides with regard to osteoclast activity. This study reports that nearly all P2-receptor-subtype mRNAs were expressed throughout human osteoclast development, and provides evidence for functional P2 receptor expression by these cells. In cultures of human osteoclasts alone, neither ATP nor UTP affected the quantity of resorption by these cells; however, in cocultures of osteoblast-like UMR-106 cells and human osteoclasts, ATP, but not UTP, greatly enhanced resorption, indicating a role for osteoblasts in mediating the proresorptive effects of ATP. Furthermore, ATP, but not UTP, elevated receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA and protein expression by UMR-106 cells. These data are consistent with observations that UMR-106 cells predominantly express P2Y(1) with low expression of P2Y(2), thereby explaining the response to ATP and not UTP, and further substantiating the involvement of osteoblasts in ATP-induced effects on osteoclasts. These results significantly advance our understanding of the role of P2 receptors in bone, and indicate that local-acting ATP may play a pivotal role in osteoclast activation at bone-resorbing sites by inducing elevated expression of RANKL.

Adenosine Triphosphate↗

PubMed1995

Osteoclast molecular phenotyping by random cDNA sequencing.

The osteoclast is a cell type that is highly specialized for its bone resorption function. In order to decipher the numerous biochemical functions of osteoclasts, a description of the gene expression profile of osteoclasts would be beneficial. We have sought to identify genes that are highly expressed in osteoclasts by partially sequencing 194 randomly chosen cDNA clones from a representative rabbit osteoclast cDNA library. Comparison to nucleic acid and protein sequence databases indicates that 135 of these cDNAs are identical to or homologous to known mammalian genes. Reverse transcription-polymerase chain reaction (RT-PCR) assays with microisolated osteoclasts were used to verify the osteoclast expression of some of these genes. Fifty-nine cDNAs, including two abundantly expressed species, have no significant similarity to the sequence databases and likely represent novel genes. The most abundant of the osteoclast expressed genes encode cofilin and the vacuolar H(+)-ATPase 16 kd subunit. Each were represented at a frequency of 4.1% of the clones in the library (95% confidence interval = 2.4-6.6%). The high expression of these gene products is consistent with the high motility of osteoclasts and their very active hydrogen ion secretion. Other abundantly expressed sequences include beta-actin (95% C.I. = 2.0-6.0%), creatine kinase B (95% C.I. = 1.2-4.9%), c-fms and ribosomal protein L18 (95% C.I. = 0.8-4.3%), and cathepsin-OC2, cyclophilin, delta-aminolevulinate synthetase, 16S mitochondrial rRNA, and two novel gene sequences (95% C.I. = 0.5-3.6%).

5-Aminolevulinate Synthetase↗

PubMed1999

Gene expression during osteoclast-like cell formation induced by antifusion regulatory protein-1/CD98/4F2 monoclonal antibodies (MAbs): c-src is selectively induced by anti-FRP-1 MAb.

Human blood monocytes can differentiate into osteoclast-like cells when they are cultured in the presence of anti-FRP-1. Messenger (mRNA) expression of markers related to osteoclasts was analyzed during differentiation of osteoclasts from monocytes. As markers related to osteoclasts, we selected cathepsin-K, carbonic anhydrase (CA) II, vacuolar H(+)-ATPase (v-ATPase), vitronectin receptor (VNR), tartrate-resistant acid phosphatase (TRAP), osteopontin (OPN), galectin-3, c-src, c-fos, and c-fms. The mRNAs other than c-src mRNA were expressed in freshly isolated monocytes or monocytes incubated with control antibody or anti-FRP-1 monoclonal antibody (MAb) for 14 days. Of these mRNAs, cathepsin-K, CA II, v-ATPase, VNR, TRAP, OPN, and c-fms mRNAs were expressed at higher levels in the osteoclast-like cells than those in monocytes cultured with control antibody. On the other hand, galectin-3 mRNA was expressed at lower levels in the osteoclast-like cells, and there was no significant difference in c-fos mRNA expression between the monocytes cultured with control antibody and anti-FRP-1 MAb. c-src mRNA could not be detected in monocytes freshly isolated or incubated with control antibody. Surprisingly, expression of c-src mRNA was induced in monocytes by anti-FRP-1 MAb and was detectable as early as 3 h after anti-FRP-1 MAb treatment, indicating that c-src is selectively induced by anti-FRP-1 MAb treatment. Furthermore, the osteoclast-like cells expressed calcitonin receptor. Receptor activator of NF-kappaB (RANK) mRNA was detectable in freshly isolated monocytes or monocytes cultured with control antibody or anti-FRP-1 MAbs. Maximal expression of RANK was observed in osteoclast-like cells. On the other hand, no receptor activator of NF-KB ligand (RANKL) mRNA was detectable in any of the samples, suggesting that anti-FRP-1 mAb can induce osteoclast-like cells from blood monocytes without RANKL.

Antibodies, Monoclonal↗

PubMed2002

The role of LFA-1 in osteoclast development induced by co-cultures of mouse bone marrow cells and MC3T3-G2/PA6 cells.

Interactions between leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) influence the development of osteoclasts. However, little is known about how these adhesion molecules are involved in the process of osteoclast development. This study evaluated the role of LFA-1 and its ligands in osteoclast development and bone resorption. Co-cultures of bone marrow cells from LFA-1-deficient mice and MC3T3-G2/PA6 (PA6) cells were cultured in the presence of 1alpha,25(OH)2D3 and dexamethasone for 7 days. The number of TRAP-positive cells that were generated by bone marrow cells from LFA-1-deficient mice was smaller than that generated by bone marrow cells from wild-type mice. In addition, the bone-resorbing activity of osteoclast-like cells that were generated from LFA-1-deficient mice was lower than that generated by osteoclast-like cells from wild-type mice. Immunofluorescence flow cytometry showed that osteoclast stromal PA6 cells expressed the cell adhesion molecules, ICAM-1 and VCAM-1. When monoclonal antibodies to mice VCAM-1, CD11b or CD18 were added separately to the co-culture system, the number of TRAP-positive cells that were generated from LFA-1-deficient mice was 20-30% smaller than that generated from wild-type mice. The formation of TRAP-positive cells from both LFA-1 deficient and wild-type mice was especially inhibited by anti-CD18 antibody, in comparison to the addition of normal IgG serum. These results suggest that LFA-1 adhesion molecules play a role in osteoclast development by affecting adhesion between stromal cells and osteoclast progenitors before the occurrence of ODF-ODF receptor signaling. CD18 appears to be a key adhesion molecule in cell-to-cell contacts during the early stage of osteoclast development.

3T3 Cells↗

PubMed1991

Osteoclastic inhibition: an action of nitric oxide not mediated by cyclic GMP.

The osteoclast is unique in its ability to resorb bone, and excessive osteoclastic activity has been implicated in osteoporosis, Paget disease of bone, rheumatoid arthritis, and the growth of metastases in bone. The activity of this cell is controlled by the main circulating inhibitor, calcitonin, in association with locally produced modulators. We show that nitric oxide (NO) may be an important member of the latter group. NO is produced by the vascular endothelium and nervous system and is involved in both neurotransmission and the regulation of blood pressure. However, our results show that the autocoid is also a potent inhibitor of osteoclast function. NO (30 microM) produced a decrease to approximately 50% of the original osteoclast spread area. Similar effects were also produced by 3-morpholinosydnonimine or sodium nitroprusside, reagents that spontaneously release NO. These shape changes were associated with a reduction of bone resorption after a 24-hr incubation of isolated osteoclasts on devitalized bone slices. NO is thought to act by stimulating guanylate cyclase, with a consequent increase in cyclic GMP, but a different mode of action is likely in the osteoclast since dibutyryl or 8-bromo cyclic GMP have no effect. It should be noted that calcitonin can produce similar changes in shape and activity but is associated with an increase in osteoclast intracellular calcium and cessation of membrane movement; neither of these is produced by NO, suggesting that its mode of action is different. The abundance of NO-producing endothelial cells in bone marrow and their proximity to osteoclasts suggests that marrow endothelial cells may play a physiological role in the regulation of osteoclastic activity.

Animals↗

PubMed1999

Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand.

A receptor that mediates osteoprotegerin ligand (OPGL)-induced osteoclast differentiation and activation has been identified via genomic analysis of a primary osteoclast precursor cell cDNA library and is identical to the tumor necrosis factor receptor (TNFR) family member RANK. The RANK mRNA was highly expressed by isolated bone marrow-derived osteoclast progenitors and by mature osteoclasts in vivo. Recombinant OPGL binds specifically to RANK expressed by transfected cell lines and purified osteoclast progenitors. Transgenic mice expressing a soluble RANK-Fc fusion protein have severe osteopetrosis because of a reduction in osteoclasts, similar to OPG transgenic mice. Recombinant RANK-Fc binds with high affinity to OPGL in vitro and blocks osteoclast differentiation and activation in vitro and in vivo. Furthermore, polyclonal Ab against the RANK extracellular domain promotes osteoclastogenesis in bone marrow cultures suggesting that RANK activation mediates the effects of OPGL on the osteoclast pathway. These data indicate that OPGL-induced osteoclastogenesis is directly mediated through RANK on osteoclast precursor cells.

Animals↗

PubMed2000

A new superoxide-generating oxidase in murine osteoclasts.

Superoxide production contributes to osteoclastic bone resorption. Evidence strongly indicates that NADPH oxidase is an enzyme system responsible for superoxide generation in osteoclasts. A membrane-bound subunit, p91, is the catalytic domain of NADPH oxidase. However, osteoclasts from p91 knockout mice still produce superoxide at a rate similar to that observed in wild type mice. This unexpected phenomenon prompted us to examine the osteoclasts for an alternative to the p91-containing oxidase. In this study, the cloning of a NADPH oxidase subunit (Nox 4) with 578 amino acids is reported. Nox 4 has 58% similarity in amino acids with the known p91 subunit of NADPH oxidase. Nox 4 is present and active in osteoclasts. Antisense oligonucleotides of Nox 4 reduced osteoclastic superoxide generation as well as resorption pit formation by osteoclasts. This new oxidase complex was present and functional in osteoclasts from p91 knockout mice, explaining the normal resorptive activity seen in the osteoclasts where no p91 is present.

Amino Acid Sequence↗

PubMed2001

Tumor necrosis factor-alpha supports the survival of osteoclasts through the activation of Akt and ERK.

Differentiated osteoclasts have a short life span. We tested various cytokines and growth factors for the effects on the survival of purified mature osteoclasts. In the absence of any added factors, osteoclasts exhibited the survival rate of less than 25% after a 24-h incubation. Among the tested factors, tumor necrosis factor-alpha (TNF-alpha) was found to increase the survival rate to approximately 80%. The TNF-alpha-enhanced survival of osteoclasts appeared to be associated with reduction in apoptosis and suppression of caspase activation. The antiapoptotic signaling pathways involved in the TNF-alpha-induced osteoclast survival were investigated. TNF-alpha treatment increased the phosphorylation of Akt in osteoclasts, which was suppressed by a phosphatidylinositol 3-kinase inhibitor LY294002 and an Src family kinase-selective inhibitor PP1. These inhibitors also attenuated the TNF-alpha stimulation of osteoclast survival. In addition an increase in the phosphorylation of ERK was observed upon TNF-alpha stimulation. PD98059, a specific inhibitor of the ERK-activating kinase MEK-1, abolished the TNF-alpha-induced ERK phosphorylation and osteoclast survival, and in these responses the involvement of Grb2 and ceramide was observed. These results suggest that TNF-alpha promotes the survival of osteoclasts by engaging the phosphatidylinositol 3-kinase Akt and MEK/ERK signaling pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2003

PYK2 autophosphorylation, but not kinase activity, is necessary for adhesion-induced association with c-Src, osteoclast spreading, and bone resorption.

Proline-rich tyrosine kinase 2 (PYK2) is the main adhesion-induced kinase in bone-resorbing osteoclasts. Previous studies have shown that ligation of alpha(v)beta(3) integrin in osteoclasts induces c-Src-dependent tyrosine phosphorylation and PYK2 activation, leading to cytoskeletal rearrangement, migration, and polarization of these cells. In this study, we examined the role of PYK2 kinase activity and its major autophosphorylation site in adhesion-dependent signaling and cytoskeletal organization during osteoclast spreading and migration. By infecting pre-fusion osteoclasts using recombinant adenovirus expressing PYK2 and its mutants, we demonstrated that mutation at the autophosphorylation site (Y402F) abolishes PYK2 association with c-Src and reduces significantly phosphorylation at tyrosines 579/580 and 881 resulting in inhibition of osteoclast spreading and bone resorption. Overexpression of the kinase-dead PYK2(K475A) mutant had no effect on cell spreading, interaction with c-Src, or the phosphorylation level of Tyr-402, Tyr-579/580, and Tyr-881 relative to PYK2(wt)-expressing cells. Taken together these findings suggest that Tyr-402 is the major docking site for c-Src and can be phosphorylated by another tyrosine kinase in osteoclasts but not in HEK293 cells. Interestingly, both PYK2(Y402F) and PYK2(K457A) translocate normally to podosomes and have no effect on macrophage colony-stimulating factor-induced osteoclast migration. Whereas PYK2(Y402F) dominant negatively blocks osteoclast spreading and bone resorption, PYK2(K457A) may function in part as an adaptor by initially recruiting c-Src to the adhesion complex, which appears to activate PYK2 by phosphorylating additional tyrosines in its regulatory and C-terminal domains. We thus concluded that phosphorylation at Tyr-402 in PYK2 is essential in the regulation of adhesion-dependent cytoskeletal organization in osteoclasts.

Animals↗

PubMed2005

P2Y6 nucleotide receptors activate NF-kappaB and increase survival of osteoclasts.

Nucleotides, released from cells during inflammation and by mechanical stimulation, act through the P2 family of nucleotide receptors. Previous studies have demonstrated the expression of P2Y1 and P2Y2 receptors in osteoclasts. The aim of this study was to determine whether osteoclast P2Y receptors signal through NF-kappaB, a key transcription factor regulating osteoclastogenesis. Immunofluorescence was used to detect the p65 subunit of NF-kappaB, which upon activation translocates from the cytosol to nuclei. Low levels of NF-kappaB activation were observed in untreated rabbit osteoclasts and in those exposed to 2-methylthio ADP (P2Y1 agonist) or ATP or UTP (P2Y2 agonists). In contrast, UDP or INS48823 (P2Y6 agonists) induced a significant increase in the number of cells exhibiting NF-kappaB activation, a process sensitive to the proteasome inhibitor lactacystin. In osteoclasts purified by micromanipulation, reverse transcription-PCR revealed the presence of P2Y1, P2Y2, and P2Y6 receptor transcripts, and application of agonists for these receptors induced the transient rise of cytosolic calcium. Treatment of rat osteoclasts with UDP or INS48823, but not 2-methylthio ADP or UTP, increased osteoclast survival. Osteoprotegerin (a decoy receptor for RANK ligand) did not significantly alter the effects of UDP on NF-kappaB localization or osteoclast survival, consistent with a direct action. Moreover, SN50 (cell-permeable peptide inhibitor of NF-kappaB) suppressed the enhancement of cell survival induced by UDP and INS48823. Our findings demonstrate the presence of functional P2Y6 receptors in osteoclasts. Thus, nucleotides, following their release at sites of inflammation and mechanical stimulation, can act through P2Y6 receptors to initiate NF-kappaB signaling and enhance osteoclast survival.

Active Transport, Cell Nucleus↗

PubMed2005

MCP-1-induced human osteoclast-like cells are tartrate-resistant acid phosphatase, NFATc1, and calcitonin receptor-positive but require receptor activator of NFkappaB ligand for bone resorption.

MCP-1 (monocyte chemotactic protein-1) is a CC chemokine that is induced by receptor activator of NFkappaB ligand (RANKL) in human osteoclasts. In the absence of RANKL, treatment of human peripheral blood mononuclear cells with macrophage colony-stimulating factor and MCP-1 resulted in tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells that are positive for calcitonin receptor (CTR) and a number of other osteoclast markers, including nuclear factor of activated t cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). Although NFATc1 was strongly induced by MCP-1 and was observed in the nucleus, MCP-1 did not permit the formation of bone-resorbing osteoclasts, although these cells had the typical TRAP(+)/CTR(+) multinuclear phenotype of osteoclasts. Despite a similar appearance to osteoclasts, RANKL treatment was required in order for TRAP(+)/CTR(+) multinuclear cells to develop bone resorption activity. The lack of bone resorption was correlated with a deficiency in expression of certain genes related to bone resorption, such as cathepsin K and MMP9. Furthermore, calcitonin blocked the MCP-1-induced formation of TRAP(+)/CTR(+) multinuclear cells as well as blocking osteoclast bone resorption activity, indicating that calcitonin acts at two stages of osteoclast differentiation. Ablation of NFATc1 in mature osteoclasts did not prevent bone resorption activity, suggesting NFATc1 is involved in cell fusion events and not bone resorption. We propose that the MCP-1-induced TRAP(+)/CTR(+) multinuclear cells represent an arrested stage in osteoclast differentiation, after NFATc1 induction and cellular fusion but prior to the development of bone resorption activity.

Acid Phosphatase↗

PubMed2006

c-Src control of chloride channel support for osteoclast HCl transport and bone resorption.

Bone degradation by osteoclasts depends upon active transport of hydrogen ions to solubilize bone mineral. This transport is supported by the parallel actions of a proton ATPase and a chloride channel located in the osteoclast ruffled membrane. We have previously identified a novel chloride channel, p62, which appears to be the avian counterpart to CLIC-5b and is expressed coincident with the appearance of acid secretion as avian osteoclasts differentiate in culture. In this article, we show that suppression of CLIC-5b in differentiating avian osteoclasts results in decreased acidification by vesicles derived from these cells and decreased ability of the cells to resorb bone. Acidification is rescued by the presence of valinomycin, consistent with a selective loss of chloride channel but not proton pump activity. Osteoclast bone resorption is known to be dependent on the expression of the tyrosine kinase, c-Src. We show that CLIC-5b from osteoclasts has affinity for both Src SH2 and SH3 domains. We find that suppression of expression of Src in developing osteoclasts results in decreased vesicular acidification, which is rescued by valinomycin, consistent with the loss of chloride conductance in the proton pump-containing vesicles. Suppression of c-Src causes no change in the steady state level of CLIC-5b expression, but does result in failure of proton pump and CLIC-5b to colocalize in cultured osteoclast precursors. We conclude that suppression of c-Src interferes with osteoclast bone resorption by disrupting functional co-localization of proton pump and CLIC-5b.

Amino Acid Sequence↗

PubMed1985

Identification of osteoclast-specific monoclonal antibodies.

Studies on the origin, identification, and characterization of osteoclasts have been difficult. This is in part due to a lack of definitive osteoclast markers and the similarity of these cells in form and function to cells of the mononuclear phagocyte system. To solve this problem, we inoculated isolated chick osteoclasts into mice to generate osteoclast-specific monoclonal antibodies. Supernatants from growth-positive hybridomas were screened by indirect immunofluorescent methods against cultured osteoclasts, monocyte-derived multinucleated giant cells, cultured monocytes, fibroblasts, and limb mesenchyme. Select hybridomas were cloned to produce 375 clones, which were analyzed as described above. Antibody from select clones was also reacted with paraffin sections of bone. In addition, two clones have been analyzed by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Antibody binding from an osteoclast-specific clone and a clone reactive with osteoclasts, giant cells, and cultured monocytes (as determined by immunohistochemical assay) was confirmed by antibody-binding and titration curves quantitated by ELISA. The above studies demonstrate that osteoclast specific antigens exist, and that osteoclasts, giant cells, and cultured monocytes share common determinants not found on other cells screened.

Animals↗

PubMed1984

Osteoclast formation from mononuclear phagocytes: role of bone-forming cells.

In a previous study, using co-cultures of embryonic bone rudiments stripped of periosteum, and mononuclear phagocytes of various sources, we found that multinucleated mineral-resorbing osteoclasts developed in vitro from radiosensitive mouse bone marrow mononuclear phagocytes (BMMP). (Burger, E. H., J. W. M. van der Meer, J. S. van de Gevel, C. W. Thesingh, and R. van Furth, 1982, J. Exp. Med. 156:1604-1614). In the present study, this co-culture technique was used to analyze the influence of bone-forming cells on osteoclast formation and bone resorption by BMMP or peritoneal exudate cells (PEC). BMMP or PEC were co-cultured with liver or dead bone, i.e., in the presence or absence of liver bone-forming cells. Mineral resorption and osteoclast formation were monitored via 45Ca release from prelabeled live or dead bone followed by histology. Osteoclasts developed from precultured BMMP as indicated by [3H]thymidine labeling, but only in live and not in dead bone. They formed readily from BMMP but only erratically, and after a longer culture period, from PEC. Macrophages from BMMP and PEC invaded live and dead bone rudiments but did not resorb the intact mineralized matrix. In contrast, ground bone powder was resorbed avidly by both cell populations, without formation of osteoclasts. We conclude that live bone-forming cells are required for osteoclast formation from progenitors. Live bone is only resorbed by osteoclasts, and not by macrophages. Osteoclast progenitors are abundant in cultures of BMMP but scarce in PEC, which makes a direct descendance of osteoclasts from mature macrophages unlikely.

Animals↗

PubMed1993

Macrophage colony-stimulating factor stimulates survival and chemotactic behavior in isolated osteoclasts.

Macrophage colony-stimulating factor (M-CSF) is known to play an important role in osteoclast formation. However, its actions on mature cells have not been fully characterized. We now report that M-CSF dramatically stimulates osteoclastic motility and spreading; osteoclasts responded to a gradient of M-CSF with orientation, and random cell polarization occurred after isotropic exposure. M-CSF also supported the survival of osteoclasts by preventing apoptosis. Paradoxically, M-CSF inhibits bone resorption by isolated osteoclasts. We found that this was effected predominantly by reduction in the number of excavations. Thus, M-CSF showed a propensity to suppress resorption through a reduction in the proportion of cells that were resorbing bone. Our data suggest that apart from the established role of M-CSF in the provision of precursors for osteoclastic induction, a major role for M-CSF in bone resorption is to enhance osteoclastic survival, migration, and chemotaxis. It seems appropriate that during these processes resorptive functions should be suppressed. We suggest that M-CSF continues to modulate osteoclastic activity once osteoclasts are on resorptive sites, through regulation of the balance between resorption and migration, such that not only the quantity, but the spatial pattern of resorption can be controlled by adjacent M-CSF-secreting cells of osteoblastic lineage.

Animals↗

PubMed2006

Estrogen directly attenuates human osteoclastogenesis, but has no effect on resorption by mature osteoclasts.

Estrogen deficiency arising with the menopause promotes marked acceleration of bone resorption, which can be restored by hormone replacement therapy. The inhibitory effects of estrogen seem to involve indirect cytokine- mediated effects via supporting bone marrow cells, but direct estrogen-receptor mediated effects on the bone-resorbing osteoclasts have also been proposed. Little information is available on whether estrogens modulate human osteoclastogenesis or merely inhibit the functional activity of osteoclasts. To clarify whether estrogens directly modulate osteoclastic activities human CD14+ monocytes were cultured in the presence of M-CSF and RANKL to induce osteoclast differentiation. Addition of 0.1-10 nM 17beta-estradiol to differentiating osteoclasts resulted in a dose-dependent reduction in tartrate resistant acid phosphatase (TRACP) activity reaching 60% at 0.1 nM. In addition, 17beta-estradiol inhibited bone resorption, as measured by the release of the C-terminal crosslinked telopeptide (CTX), by 60% at 0.1 nM, but had no effect on the overall cell viability. In contrast to the results obtained with differentiating osteoclasts, addition of 17beta-estradiol (0.001-10 nM) to mature osteoclasts did not affect bone resorption or TRACP activity. We investigated expression of the estrogen receptors, using immunocytochemistry and Western blotting. We found that ER-alpha is expressed in osteoclast precursors, whereas ER- beta is expressed at all stages, indicating that the inhibitory effect of estrogen on osteoclastogenesis is mediated by ER-alpha for the major part. In conclusion, these results suggest that the in vivo effects of estrogen are mediated by reduction of osteoclastogenesis rather than direct inhibition of the resorptive activity of mature osteoclasts.

Blotting, Western↗

PubMed2004

Apatite-mediated actin dynamics in resorbing osteoclasts.

The actin cytoskeleton is essential for osteoclasts main function, bone resorption. Two different organizations of actin have been described in osteoclasts, the podosomes belt corresponding to numerous F-actin columns arranged at the cell periphery, and the sealing zone defined as a unique large band of actin. To compare the role of these two different actin organizations, we imaged osteoclasts on various substrata: glass, dentin, and apatite. Using primary osteoclasts expressing GFP-actin, we found that podosome belts and sealing zones, both very dynamic actin structures, were present in mature osteoclasts; podosome belts were observed only in spread osteoclasts adhering onto glass, whereas sealing zone were seen in apico-basal polarized osteoclasts adherent on mineralized matrix. Dynamic observations of several resorption cycles of osteoclasts seeded on apatite revealed that 1) podosomes do not fuse together to form the sealing zone; 2) osteoclasts alternate successive stationary polarized resorption phases with a sealing zone and migration, nonresorption phases without any specific actin structure; and 3) apatite itself promotes sealing zone formation though c-src and Rho signaling. Finally, our work suggests that apatite-mediated sealing zone formation is dependent on both c-src and Rho whereas apico-basal polarization requires only Rho.

Actins↗

PubMed2003

Osteoclast differentiation at growth plate cartilage-trabecular bone junction in newborn rat femur.

Using 3-day-old newborn rats, we examined the differentiation processes of osteoclasts associated with the destruction of the femoral growth plate cartilage and primary trabecular bone. In the growth plate cartilage, thin mineralized areas were detected solely in the longitudinal septal cartilage matrix in the hypertrophic zone, but the transverse septal cartilage matrix between adjacent chondrocytic lacunae within a row of chondrocytes remained unmineralized. The longitudinal septal cartilage between adjacent rows of chondrocytes appeared to persist, forming the walls of opened lacunar canals. Consistent with the removal of the transverse septal cartilage matrix, the longitudinal canals of opened chondrocytic lacunae were deeply invaded by capillary vessels, mononuclear cells and multinucleated pre-osteoclasts lacking a ruffled border. CD34-positive endothelial cells of capillary vessels deeply penetrated into the transverse septal cartilage matrix facing the medullary cavity and the opened chondrocytic lacunae. ED1-positive monocytes/macrophages were distributed at the chondro-osseous junction, but they were distant from the erosive front of the transverse septa. Tartrate-resistant acid phosphatase-positive multinucleated pre-osteoclasts lacking a ruffled border and differentiated osteoclasts with a ruffled border were localized mainly at two locations: the chondro-osseous junction and the growth front of primary bone trabeculae. Osteoclasts were located on the type-I collagen-positive bone trabeculae close to the growth plate, but they appeared to be distant from the type-II collagen-positive cartilage matrix. Even within opened chondrocytic lacunae, when osteoclasts were distant from the cartilage and bone matrix, they lacked polarized cytoplasmic organization and a ruffled border. The osteoclasts located in the remaining septal cartilage also exhibited neither a ruffled border nor a clear zone. Osteoclasts with a prominent ruffled border and clear zone were located in bone matrix covering the remaining septal cartilage. These results suggest that osteoclasts require hydroxyapatite crystals and bone matrix constituents for ruffled border formation and are not involved in resorption of the unmineralized transverse and mineralized longitudinal septal cartilage without covering bone matrix at the chondro-osseous junction.

Animals↗