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PubMed2002

Osteoclasts are essential for TNF-alpha-mediated joint destruction.

The detailed cellular and molecular mechanisms leading to joint destruction in rheumatoid arthritis, a disease driven by proinflammatory cytokines, are still unknown. To address the question of whether osteoclasts play a pivotal role in this process, transgenic mice that express human TNF (hTNFtg) and that develop a severe and destructive arthritis were crossed with osteopetrotic, c-fos-deficient mice (c-fos(-/-)) completely lacking osteoclasts. The resulting mutant mice (c-fos(-/-)hTNFtg) developed a TNF-dependent arthritis in the absence of osteoclasts. All clinical features of arthritis, such as paw swelling and reduction of grip strength, progressed equally in both groups. Histological evaluation of joint sections revealed no difference in the extent of synovial inflammation, its cellular composition (except for the lack of osteoclasts), and the expression of matrix metalloprotein-ase-3 (MMP-3) and MMP-13. In addition, cartilage damage, proteoglycan loss, and MMP-3, -9, and -13 expression in chondrocytes were similar in hTNFtg and c-fos(-/-)hTNFtg mice. However, despite the presence of severe inflammatory changes, c-fos(-/-)hTNFtg mice were fully protected against bone destruction. These data reveal that TNF-dependent bone erosion is mediated by osteoclasts and that the absence of osteoclasts alters TNF-mediated arthritis from a destructive to a nondestructive arthritis. Therefore, in addition to the use of anti-inflammatory therapies, osteoclast inhibition could be beneficial for the treatment of rheumatoid arthritis.

Animals↗

PubMed2005

FHL2 inhibits the activated osteoclast in a TRAF6-dependent manner.

TNF receptor-associated factor 6 (TRAF6) associates with the cytoplasmic domain of receptor activator of NF-kappaB (RANK). This event is central to normal osteoclastogenesis. We discovered that TRAF6 also interacts with FHL2 (four and a half LIM domain 2), a LIM domain--only protein that functions as a transcriptional coactivator or corepressor in a cell-type--specific manner. FHL2 mRNA and protein are undetectable in marrow macrophages and increase pari passu with osteoclast differentiation in vitro. FHL2 inhibits TRAF6-induced NF-kappaB activity in wild-type osteoclast precursors and, in keeping with its role as a suppressor of TRAF6-mediated RANK signaling, TRAF6/RANK association is enhanced in FHL2-/- osteoclasts. FHL2 overexpression delays RANK ligand-induced (RANKL-induced) osteoclast formation and cytoskeletal organization. Interestingly, osteoclast-residing FHL2 is not detectable in naive wild-type mice, in vivo, but is abundant in those treated with RANKL and following induction of inflammatory arthritis. Reflecting increased RANKL sensitivity, osteoclasts generated from FHL2-/- mice reach maturation and optimally organize their cytoskeleton earlier than their wild-type counterparts. As a consequence, FHL2-/- osteoclasts are hyperresorptive, and mice lacking the protein undergo enhanced RANKL and inflammatory arthritis-stimulated bone loss. FHL2 is, therefore, an antiosteoclastogenic molecule exerting its effect by attenuating TRAF6-mediated RANK signaling.

Animals↗

PubMed2000

Mice lacking beta3 integrins are osteosclerotic because of dysfunctional osteoclasts.

Osteoclasts express the alphavbeta3 integrin, an adhesion receptor that has been implicated in bone resorption and that is therefore a potential therapeutic target. To assess the role of this heterodimer in skeletal development in vivo, we engineered mice in which the gene for the beta3 integrin subunit was deleted. Bone marrow macrophages derived from these mutants differentiate in vitro into numerous osteoclasts, thus establishing that alphavbeta3 is not necessary for osteoclast recruitment. Furthermore, the closely related integrin, alphavbeta5, does not substitute for alphavbeta3 during cytokine stimulation or authentic osteoclastogenesis. beta3 knockout mice, but not their heterozygous littermates, develop histologically and radiographically evident osteosclerosis with age. Despite their increased bone mass, beta3-null mice contain 3.5-fold more osteoclasts than do heterozygotes. These mutant osteoclasts are, however, dysfunctional, as evidenced by their reduced ability to resorb whale dentin in vitro and the significant hypocalcemia seen in the knockout mice. The resorptive defect in beta3-deficient osteoclasts may reflect absence of matrix-derived intracellular signals, since their cytoskeleton is distinctly abnormal and they fail to spread in vitro, to form actin rings ex vivo, or to form normal ruffled membranes in vivo. Thus, although it is not required for osteoclastogenesis, the integrin alphavbeta3 is essential for normal osteoclast function.

Animals↗

PubMed1990

Endocytosis in odontoclasts and osteoclasts using microperoxidase as a tracer.

Microperoxidase (MP, a peptide tracer) was intravenously injected into rats after six days of tooth movement by the Waldo method. Bone resorption was seen along the distal bone surface of the inter-radicular septum of the upper first molar, and tooth root resorption occurred along the mesial and distal surfaces of the distal root of the same molar. Odontoclasts were smaller in size and numbers than osteoclasts, but had the same organelles [multiple nuclei, ruffled borders (RB), clear zones, vacuoles, mitochondria, and specific granules] as osteoclasts. MP was deposited on the resorbed area through the clear zone and was transported into the vacuoles along the channels of the RB. The uptake of MP by odontoclasts was small, compared with that by osteoclasts. Collagen fibrils were found in the channels of the RB but were not detected in the vacuoles. Instead, filament-like structures were seen in the vacuoles and were located very near the collagen fibrils in the channels. Fibroblasts outside the resorbed lacunae had endocytosed collagen. In contrast, some cells close to odontoclasts (osteoclasts) in the dentin (bone)-resorbed lacunae had not endocytosed any collagen fibrils. These findings suggest (1) that odontoclasts resorb the dentin or cementum just as osteoclasts do in bone resorption, (2) that the resorbed area connects with the extracellular spaces of the odontoclasts or osteoclasts, and (3) that the organic components (e.g., collagen fibrils) of dentin or cementum are endocytosed through the channels of the RB in the same manner as that for MP. However, the endocytotic ability of odontoclasts appears inferior to that of osteoclasts.

Animals↗

PubMed2004

Monokine induced by interferon-gamma is induced by receptor activator of nuclear factor kappa B ligand and is involved in osteoclast adhesion and migration.

Bone remodeling is accompanied by the differentiation of osteoclasts from the monocyte/macrophage lineage of hematopoietic cells. The osteoclast differentiation process requires receptor activator of nuclear factor kappa B (NF-kappa B) ligand (RANKL), which causes complex changes in the expression of various genes. In a cDNA microarray study to identify genes targeted by RANKL, we found that monokine induced by the interferon-gamma (IFN-gamma) (MIG) gene was up-regulated in osteoclast precursor cells. The increase in MIG expression by RANKL was confirmed by reverse transcription-polymerase chain reaction and Western blot analysis. RANKL induction of MIG required the activity of NF-kappa B, whose binding site is present in the MIG promoter. MIG induction by RANKL was also dependent on p38 mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 1 (STAT1). RANKL stimulated the phosphorylation of Ser727 of STAT1, which required p38 activity. MIG secreted on RANKL treatment could stimulate the migration and adhesion of osteoclast precursors and osteoclasts that were primed to express CXCR3, the MIG receptor, by macrophage-colony-stimulating factor (M-CSF). Therefore, we provide the first evidence demonstrating that RANKL stimulates the serine phosphorylation of STAT1 through the p38 MAPK pathway, causing MIG gene transcription and secretion, which may have a role in recruiting CXCR3-positive osteoclast precursors and osteoclasts to bone remodeling or inflammatory sites.

Animals↗

PubMed2005

Chemokine and chemokine receptor expression during colony stimulating factor-1-induced osteoclast differentiation in the toothless osteopetrotic rat: a key role for CCL9 (MIP-1gamma) in osteoclastogenesis in vivo and in vitro.

Osteoclasts differentiate from hematopoietic precursors under systemic and local controls. Chemokines and receptors direct leukocyte traffic throughout the body and may help regulate site-specific bone resorption. We investigated bone gene expression in vivo during rapid osteoclast differentiation induced by colony-stimulating factor 1 (CSF-1) in Csf1-null toothless (tl/tl) rats. Long-bone RNA from CSF-1-treated tl/tl rats was analyzed by high-density microarray over a time course. TRAP (tartrate-resistant acid phosphatase)-positive osteoclasts appeared on day 2, peaked on day 4, and decreased slightly on day 6, as marrow space was expanding. TRAP and cathepsin K mRNA paralleled the cell counts. We examined all chemokine and receptor mRNAs on the arrays. CCL9 was strongly induced and peaked on day 2, as did its receptor, CCR1, and regulatory receptors c-Fms (CSF-1 receptor) and RANK (receptor activator of nuclear factor kappaB). Other chemokines and receptors showed little or no significant changes. In situ hybridization and immunohistochemistry revealed CCL9 in small, immature osteoclasts on day 2 and in mature cells at later times. Anti-CCL9 antibody inhibited osteoclast differentiation in culture and significantly suppressed the osteoclast response in CSF-1-treated tl/tl rats. While various chemokines have been implicated in osteoclastogenesis in vitro, this first systematic analysis of chemokines and receptors during osteoclast differentiation in vivo highlights the key role of CCL9 in this process.

Animals↗

PubMed2006

Endostatin inhibits VEGF-A induced osteoclastic bone resorption in vitro.

BACKGROUND: Endostatin is a C-terminal fragment of collagen XVIII which is a component of basement membranes with the structural properties of both collagens and proteoglycans. Endostatin has a major role in angiogenesis which is intimately associated with bone development and remodeling. Signaling between the endothelial cells and the bone cells, for example, may have a role in recruitment of osteoclastic precursor cells. Our study aims at exploring a possibility that endostatin, either as a part of basement membrane or as a soluble molecule, may control osteoclastogenesis and osteoclastic bone resorption in vitro. METHODS: Rat pit formation assay was employed in order to examine the effect of endostatin alone or in combination with vascular endothelial growth factor-A (VEGF-A) on bone resorption in vitro. Effect of these agents on osteoclast differentiation in vitro was also tested. Osteoclastogenesis and the number of osteoclasts were followed by tartrate resistant acid phosphatase (TRACP) staining and resorption was evaluated by measuring the area of excavated pits. RESULTS: Endostatin inhibited the VEGF-A stimulated osteoclastic bone resorption, whereas endostatin alone had no effect on the basal resorption level in the absence of VEGF-A. In addition, endostatin could inhibit osteoclast differentiation in vitro independent of VEGF-A. CONCLUSION: Our in vitro data indicate that collagen XVIII/endostatin can suppress VEGF-A induced osteoclastic bone resorption to the basal level. Osteoclastogenesis is also inhibited by endostatin. The regulatory effect of endostatin, however, is not critical since endostatin alone does not modify the basal bone resorption.

Acid Phosphatase↗

PubMed2006

Inhibitory effect of ribbon-type NF-kappaB decoy oligodeoxynucleotides on osteoclast induction and activity in vitro and in vivo.

In this study we examined the effect of ribbon-type (circular-type) NF-kappaB decoy oligodeoxynucleotides (RNODN) on osteoclast induction and activity. We extracted bone marrow cells from the femurs of rats and incubated non-adherent cells with receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). First, transfer efficiency into osteoclasts and their precursors, resistance to exonuclease, and binding activity of decoy to NF-kappaB were examined. Next, to examine the effect of RNODN on osteoclast induction and activity, osteoclast differentiation and pit formation assays were performed. RNODN were injected into the ankle joints of rats with collagen-induced arthritis. Joint destruction and osteoclast activity were examined by histological study. The resistance of RNODN to exonuclease and their binding activity on NF-kappaB were both greater than those of phosphorothionated NF-kappaB decoy oligodeoxynucleotides. The absolute number of multinucleate cells scoring positive for tartrate-resistant acid phosphatase was significantly decreased in the RNODN-treated group. The average calcified matrix resorbed area was significantly decreased in the RNODN-treated group. Histological study showed marked suppression of joint destruction and osteoclast activity by intra-articular injection of RNODN. These results suggest the inhibitory effect of RNODN on the induction and activity of osteoclasts. Direct intra-articular injection of RNODN into the joints may be an effective strategy for the treatment of arthritis.

Animals↗

PubMed2005

Mathematical model of paracrine interactions between osteoclasts and osteoblasts predicts anabolic action of parathyroid hormone on bone.

To restore falling plasma calcium levels, PTH promotes calcium liberation from bone. PTH targets bone-forming cells, osteoblasts, to increase expression of the cytokine receptor activator of nuclear factor kappaB ligand (RANKL), which then stimulates osteoclastic bone resorption. Intriguingly, whereas continuous administration of PTH decreases bone mass, intermittent PTH has an anabolic effect on bone, which was proposed to arise from direct effects of PTH on osteoblastic bone formation. However, antiresorptive therapies impair the ability of PTH to increase bone mass, indicating a complex role for osteoclasts in the process. We developed a mathematical model that describes the actions of PTH at a single site of bone remodeling, where osteoclasts and osteoblasts are regulated by local autocrine and paracrine factors. It was assumed that PTH acts only to increase the production of RANKL by osteoblasts. As a result, PTH stimulated osteoclasts upon application, followed by compensatory osteoblast activation due to the coupling of osteoblasts to osteoclasts through local paracrine factors. Continuous PTH administration resulted in net bone loss, because bone resorption preceded bone formation at all times. In contrast, over a wide range of model parameters, short application of PTH resulted in a net increase in bone mass, because osteoclasts were rapidly removed upon PTH withdrawal, enabling osteoblasts to rebuild the bone. In excellent agreement with experimental findings, increase in the rate of osteoclast death abolished the anabolic effect of PTH on bone. This study presents an original concept for the regulation of bone remodeling by PTH, currently the only approved anabolic treatment for osteoporosis.

Animals↗

PubMed2005

Cells of the osteoclast lineage as mediators of the anabolic actions of parathyroid hormone in bone.

PTH is an anabolic agent used to treat osteoporosis, but its mechanisms of action are unclear. This study elucidated target cells and mechanisms for anabolic actions of PTH in mice during bone growth. Mice with c-fos ablation are osteopetrotic and lack an anabolic response to PTH. In this study, there were no alterations in PTH-regulated osteoblast differentiation or proliferation in vitro in cells from c-fos -/- mice compared with +/+; hence, the impact of osteoclastic cells was further investigated. A novel transplant model was used to rescue the osteopetrotic defect of c-fos ablation. Vertebral bodies (vossicles) from c-fos -/- and +/+ mice were implanted into athymic hosts, and the c-fos -/- osteoclast defect was rescued. PTH treatment to vossicle-bearing mice increased 5-bromo-2'-deoxyuridine (BrdU) positivity in the bone marrow and increased bone area regardless of the vossicle genotype. To inhibit recruitment of osteoclast precursors to wild-type vossicles, stromal derived factor-1 signaling was blocked, which blunted the PTH anabolic response. Treating mice with osteoprotegerin to inhibit osteoclast differentiation also blocked the anabolic action of PTH. In contrast, using c-src mutant mice with a late osteoclast differentiation defect did not hinder the anabolic action, suggesting key target cells reside in the intermediately differentiated osteoclast population in the bone marrow. These results indicate that c-fos in osteoblasts is not critical for PTH action but that cells of the osteoclast lineage are intermediate targets for the anabolic action of PTH.

Anabolic Agents↗

PubMed1990

Inhibitory effects of the bone-derived growth factors osteoinductive factor and transforming growth factor-beta on isolated osteoclasts.

Demineralized bone matrix contains a number of growth factors for osteoblast-like cells. Two of these, the novel glycoprotein osteoinductive factor (OIF) and transforming growth factor-beta (TGF beta), act together to cause ectopic bone formation in vivo. Since OIF, like TGF beta, is likely released from bone when the matrix is resorbed, we examined the effects of homogeneous OIF and TGF beta on osteoclast function. Osteoclast function was tested in isolated avian osteoclasts and was measured in terms of tartrate-resistant acid phosphatase (TRAP) activity, oxygen-derived free radical production, and formation of characteristic resorption lacunae on slices of sperm whale dentine. OIF (50-100 ng/ml) inhibited the capacity of these osteoclasts to form lacunae whether assessed by the number of excavations per slice or by the total area resorbed. OIF (10-100 ng/ml) or TGF beta (10-20 ng/ml) caused a decrease in TRAP activity as well as a reduction in oxygen-derived free radical generation detected by nitroblue tetrazolium staining. TGF beta had no effect on the resorption capacity of isolated osteoclasts in concentrations that inhibited TRAP activity and nitroblue tetrazolium staining. These results suggest that growth regulatory factors, such as OIF and TGF beta, released during the resorption of bone may be endogenous inhibitors of continued osteoclastic activity. This cessation of osteoclast activity may be an essential preliminary step to the new bone formation that occurs at resorption sites during bone remodeling.

Acid Phosphatase↗

PubMed1991

Generation of avian cells resembling osteoclasts from mononuclear phagocytes.

Several lines of indirect evidence suggest that a monocyte family precursor gives rise to the osteoclast, although this hypothesis is controversial. Starting with a uniform population of nonspecific esterase positive, tartrate-sensitive, acid phosphatase-producing, mannose receptor-bearing mononuclear cells, prepared from dispersed marrow of calcium-deprived laying hens by cell density separation and selective cellular adherence, we generated multinucleated cells in vitro. When cultured with devitalized bone, these cells show, by electron microscopy, the characteristic osteoclast morphology in that they are mitochondria-rich, multinucleated, and, most importantly, develop characteristic ruffled membranes at the matrix attachment site. Moreover, as documented by scanning electron microscopy, these cells pit bone slices in a manner identical to freshly isolated osteoclasts. In addition, isoenzymes of acid phosphatase from generated osteoclasts, separated by 7.5% polyacrylamide gel electrophoresis at pH 4, are identical to those of mature osteoclasts in migration pattern and tartrate resistance, although the precursor cells from which the osteoclasts are generated produce an entirely different isoenzyme, which is tartrate-sensitive and migrates less rapidly at pH 4. The fused cells also exhibit a cAMP response to prostaglandin E2. Therefore, osteoclast-like cells can be derived by in vitro culture of a marrow-derived monocyte cell population.

Animals↗

PubMed1992

12-O-tetradecanoylphorbol-13-acetate inhibits osteoclast-like cell differentiation in rat bone marrow cultures by inducing macrophage polykaryons.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) at 10(-9) M stimulated the formation of osteoclast-like multinucleated cells (MNCs) in the presence of 1 alpha,25-dihydroxyvitamin D3 in rat bone marrow cultures. However, at 10(-7) M, it clearly inhibited 1 alpha,25-dihydroxyvitamin D3-dependent osteoclast-like MNC formation at 6 days of culture. In cultures treated with 10(-7) M TPA, numerous MNCs that lack the marker enzyme tartrate-resistant acid phosphatase (TRAP) were formed. These TRAP-negative MNCs had neither receptors for calcitonin nor dentine-resorbing activity. The reactivity of the cells against antirat macrophage antibodies was completely different from that of authentic osteoclasts. These data suggest that TRAP-negative MNCs formed in the presence of 10(-7) M TPA are macrophage polykaryons. Time-course studies showed that 10(-7) M TPA stimulated osteoclast-like MNC formation at 4 days of culture, but these osteoclast-like MNCs were converted to TRAP-negative MNCs. Furthermore, 1-(5-isoquinolinyl-sulfonyl)2-methylpiperazine (H-7), a protein kinase-C inhibitor, inhibited osteoclast-like MNC formation in a dose-dependent fashion. These results suggest that activation of protein kinase-C may play a role in osteoclast differentiation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1996

A role for CD8+ T lymphocytes in osteoclast differentiation in vitro.

Bone loss associated with chronic inflammatory diseases has been attributed to the release of cytokines from T lymphocytes. However, the role of T lymphocyte subsets in the mediation of osteoclast activity has not been extensively studied. Cocultures of murine bone marrow and BALC cells (murine calvarial-derived cell line) were used to study osteoclast differentiation. Murine marrow was left intact or depleted of cells expressing the CD8 or CD4 antigen by immunomagnetic separation and then cocultured with BALC cells in the presence or absence of 1,25-(OH)2D3. Depleting bone marrow of CD4-positive (CD4+) cells did not affect osteoclast differentiation (formation of tartrate-resistant acid phosphatase positive cells with three or more nuclei). However, depletion of CD8-positive (CD8+) cells resulted in a 40% increase in the number of osteoclasts formed. Addition of CD8+ cells to CD8+ cell depleted cocultures via Transwells abolished the stimulatory effects on osteoclast differentiation resulting from CD8+ cell depletion. Neutralizing antibodies to interleukin-4 and transforming growth factor-beta did not affect osteoclast differentiation in these cultures. These findings suggest that CD8+ cells may be involved in the regulation of osteoclast differentiation and that this effect is not mediated by interleukin-4 or transforming growth factor-beta.

Animals↗

PubMed1998

Osteoclasts from human giant cell tumors of bone lack estrogen receptors.

Although estrogen is important in human skeletal homeostasis, the major target cell in bone is unknown. Estrogen receptors (ER) have been demonstrated in osteoblasts and bone marrow stromal cells, but their presence in osteoclasts remains controversial because completely pure preparations have not been available. We have examined expression of ER-alpha and ER-beta messenger RNA (mRNA) by RT-PCR in samples from human giant cell tumor of bone (GCT), including: whole tumor, cultured mononuclear cells, and a pure osteoclast population obtained by microisolation. Whole tumor expressed both ER-alpha and calcitonin receptor (CTR) mRNA and apparently lower levels of ER-beta mRNA. Passaged cultures of tumor mononuclear stromal cells also expressed ER-alpha and low ER-beta but not CTR mRNA. In pure preparations of microisolated osteoclasts, expression of ER-alpha or ER-beta mRNA was not detected, whereas expression of CTR mRNA was readily identified. Microisolated GCT mononuclear cells expressed ER-alpha, but no detectable CTR mRNA. Fluorescence in situ hybridization (FISH) using an ER-alpha riboprobe demonstrated strong signal in the mononuclear cells but multinucleated osteoclasts showed no detectable signal. In contrast, CTR mRNA was detected in multinucleated osteoclasts but not in stromal-like tumor cells by FISH. 17Beta-estradiol consistently showed no effect on bone resorbing activity of osteoclasts from GCT cultured on cortical bone, although calcitonin was a potent inhibitor. These findings indicate that significant expression of ER does not occur in osteoclasts derived from human GCT and suggest that estrogen effects are mediated by other cells of the bone environment.

Bone Neoplasms↗

PubMed1983

The sensitivity of isolated osteoclasts to morphological transformation by calcitonin.

We recently developed a technique for isolating osteoclasts from other bone cells which allows the direct effect of hormones on these cells to be assessed. We found that calcitonin (CT), which inhibits osteoclastic bone resorption, abolished cytoplasmic movement in these cells. In this paper we use the shape-change which accompanies immotility to define the sensitivity of osteoclasts to human and salmon CT. Concentrations of human CT above 62 pg/ml and salmon CT above 3 pg/ml regularly induced the shape-change in osteoclasts which corresponds to complete inhibition of cytoplasmic motility. Morphological transformation of osteoclasts by such low concentrations implies that CT may inhibit bone resorption by inhibiting the motility of the bone-resorptive cell. Even relatively high concentrations of other hormones had no effect on the sensitivity of osteoclasts to CT. This suggests that the response of isolated osteoclasts to CT is sufficiently specific and sensitive to be used as a bioassay, and we used the response to compare the biological potency of several samples of known immunoassayable CT content.

1-Methyl-3-isobutylxanthine↗

PubMed1986

The effect of human calcitonin on the cytoplasmic spreading of rat osteoclasts.

We previously found that calcitonin (CT), which inhibits osteoclastic bone resorption, abolished the cytoplasmic motility of isolated osteoclasts. The transition from motility to immotility was accompanied by a characteristic change in cell shape which coincided with the loss of pseudopodial ruffling activity and gradual cytoplasmic retraction. In this report we used computer-assisted morphometry to quantify the reduction in cytoplasmic spreading induced by human CT (hCT). Osteoclasts, isolated from neonatal rat long bones, were allowed to spread on a plastic surface for 120 min. The outlines of six osteoclasts were recorded on a time-lapse video recorder. hCT, other hormones, or plasma samples were then added. The outlines of the same six osteoclasts were recorded after incubation, and the area covered by the cells after incubation was calculated as a percentage of the area covered by the same cells before hormone addition. Osteoclastic spreading was reduced by hCT in a dose-dependent manner and was significantly decreased by concentrations of 2 pg/ml and above. High concentrations of PTH and 1,25-dihydroxyvitamin D3 had no effect on osteoclasts. This technique is a sensitive and quantitative assay of hCT concentration which is unaffected by the presence of other calcium-regulating hormones likely to be present in plasma.

Aged↗

PubMed2002

Osteoclast spreading kinetics are correlated with an oscillatory activation of a calcium-dependent potassium current.

Cell movement and spreading involve calcium-dependent processes and ionic channel activation. During bone resorption, osteoclasts alternate between spread, motile and resorptive phases. We investigated whether the electrical membrane properties of osteoclasts were linked to their membrane morphological changes. Rabbit osteoclasts were recorded by time-lapse videomicroscopy performed simultaneously with patch-clamp whole cell and single channel recordings. Original image analysis methods were developed and used to demonstrate for the first time an oscillatory activation of a spontaneous membrane current in osteoclasts, which is directly correlated to the membrane movement rate. This current was identified as a calcium-dependent potassium current (IK(Ca)) that is sensitive to both charybdotoxin and apamin and was generated by a channel with unitary conductance of approximately 25+/-2 pS. Blockade of this current also decreased osteoclast spreading and inhibited bone resorption in vitro, demonstrating a physiological role for this current in osteoclast activity. These results establish for the first time a temporal correlation between lamellipodia formation kinetics and spontaneous peaks of IK(Ca), which are both involved in the control of osteoclast spreading and bone resorption.

Animals↗