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PubMed2005

Dynamin forms a Src kinase-sensitive complex with Cbl and regulates podosomes and osteoclast activity.

Podosomes are highly dynamic actin-containing adhesion structures found in osteoclasts, macrophages, and Rous sarcoma virus (RSV)-transformed fibroblasts. After integrin engagement, Pyk2 recruits Src and the adaptor protein Cbl, forming a molecular signaling complex that is critical for cell migration, and deletion of any molecule in this complex disrupts podosome ring formation and/or decreases osteoclast migration. Dynamin, a GTPase essential for endocytosis, is also involved in actin cytoskeleton remodeling and is localized to podosomes where it has a role in actin turnover. We found that dynamin colocalizes with Cbl in the actin-rich podosome belt of osteoclasts and that dynamin forms a complex with Cbl in osteoclasts and when overexpressed in 293VnR or SYF cells. The association of dynamin with Cbl in osteoclasts was decreased by Src tyrosine kinase activity and we found that destabilization of the dynamin-Cbl complex involves the recruitment of Src through the proline-rich domain of Cbl. Overexpression of dynamin increased osteoclast bone resorbing activity and migration, whereas overexpression of dynK44A decreased osteoclast resorption and migration. These studies suggest that dynamin, Cbl, and Src coordinately participate in signaling complexes that are important in the assembly and remodeling of the actin cytoskeleton, leading to changes in osteoclast adhesion, migration, and resorption.

Actins↗

PubMed2003

Extremely high expression of beta-actin mRNA in osteoclasts resorbing alveolar bone located at the distal area of the developing molar tooth germ in newborn rats.

Expression of beta-actin is widely utilized as an internal control of mRNA expression in various cells. Here we show evidence that the expression level of beta-actin mRNA in osteoclasts significantly differs in its intensity according to the position in bone tissues. By use of in situ hybridization, we obtained clear data showing that osteoclasts facing the distal part of a developing molar tooth germ expressed extremely high levels of beta-actin mRNA in comparison with other osteoclasts observed in the mandibular bone surface. No signal was detected when beta-nerve growth factor transcripts were observed. Electron microscopic analysis revealed that osteoclasts localized in these areas were functionally active, estimated from the expression of high levels of the osteoclast membrane antigen, Kat1 antigen, which associated with active osteoclasts. These data suggest that osteoclasts expressing extremely high levels of beta-actin are highly related to active osteoclasts induced by the mechanical stress caused by physiological movement of molar tooth germ towards distal directions in the developing mandible.

Actins↗

PubMed2003

Cathepsins in the osteoclast.

The mechanism by which bone collagen and other organic components are degraded by the osteoclast during osteoclastic bone resorption was unclear until the 1980s. Studies conducted since the early 1990s have identified lysosomal proteases, mainly cathepsins that are active at low pH, involved in osteoclastic bone resorption. Several cathepsins, such as cathepsins C, D, B, E, G and L, were initially demonstrated to take part in the degradation of organic bone matrix in osteoclasts. Cathepsin K, which has high proteolytic activity and localizes primarily in osteoclasts, was discovered in 1995. This first tissue-specific cathepsin was associated with pycnodysostosis, a genetic disorder observable as an osteopetrotic phenotype in cathepsin K-deficient mice. Cystatin C, an endogenous inhibitor of cysteine proteases, regulates the activity of cathepsin K. However, detailed morphological observations suggest that the organic bone matrix is degraded by not only cathepsin K, but also by matrix metalloproteinases or other cathepsins. The osteoclast possesses a unique endocytotic/exocytotic structure and each cathepsin is specifically localized in the osteoclast, which implies that each cathepsin contributes cooperatively to the process of osteoclastic bone resorption. Further studies may clarify the regulation of cathepsin activities and the roles of cathepsins during bone remodelling.

Animals↗

PubMed2003

Disordered osteoclast formation and function in a CD38 (ADP-ribosyl cyclase)-deficient mouse establishes an essential role for CD38 in bone resorption.

We have evaluated the role of the ADP-ribosyl cyclase, CD38, in bone remodeling, a process by which the skeleton is being renewed constantly through the coordinated activity of osteoclasts and osteoblasts. CD38 catalyzes the cyclization of its substrate, NAD+, to the Ca2+-releasing second messenger, cyclic ADP-ribose (cADPr). We have shown previously that CD38 is expressed both in osteoblasts and osteoclasts. Its activation in the osteoclast triggers Ca2+ release through ryanodine receptors (RyRs), stimulation of interleukin-6 (IL-6), and an inhibition of bone resorption. Here, we have examined the consequences of deleting the CD38 gene in mice on skeletal remodeling. We report that CD38-/- mice displayed a markedly reduced bone mineral density (BMD) at the femur, tibia, and lumbar spine at 3 months and at the lumbar spine at 4 months, with full normalization of the BMD at all sites at 5 months. The osteoporosis at 3 months was accompanied by a reduction in primary spongiosa and increased osteoclast surfaces on histomorphometric analysis. Hematopoetic stem cells isolated ex vivo from CD38-/- mice showed a dramatic approximately fourfold increase in osteoclast formation in response to incubation for 6 days with RANK-L and M-CSF. The osteoclasts so formed in these cultures showed a approximately 2.5-fold increase in resorptive activity compared with wild-type cells. However, when adherent bone marrow stromal cells were allowed to mature into alkaline phosphatase-positive colony-forming units (CFU-Fs), those derived from CD38-/- mice showed a significant reduction in differentiation compared with wild-type cells. Real-time RT-PCR on mRNA isolated from osteoclasts at day 6 showed a significant reduction in IL-6 and IL-6 receptor mRNA, together with significant decreases in the expression of all calcineurin A isoforms, alpha, beta, and gamma. These findings establish a critical role for CD38 in osteoclast formation and bone resorption. We speculate that CD38 functions as a cellular NAD+ "sensor," particularly during periods of active motility and secretion.

ADP-ribosyl Cyclase↗

PubMed2005

Functional osteoclast-like transformation of cultured human myeloma cell lines.

Hyperactive osteoclastogenesis is a hallmark of multiple myeloma, a B cell neoplasia homing to bone marrow and resulting in multiple osteolytic lesions and skeleton devastation. We provide evidence that myeloma cells can themselves act as osteoclasts in vitro. By extending standard cultures of U-266 and MCC-2 myeloma cell lines, we found that subsets of adherent cells also expressed the osteoclast phenotype, including multinuclear morphology, cytoplasmic tartrate-resistant acid phosphatase, the calcitonin receptor and a specific osteoclast antigen. These subsets resorbed bone substrates by producing osteoclast enzymes as well as the characteristic redistribution of F-actin in their cytoskeleton, thus forming the sealing zone that is adopted by adherent osteoclasts to generate the acidified environment essential for bone resorption. Neither the phenotype nor the functional properties of osteoclasts were detected in parental non-adherent cells. In adherent cultures osteoclastogenesis was associated with deregulated expression of both receptor activator of nuclear transcription factor (NF)-kappaB (RANK) and its ligand RANK-L, which triggers cell maturation in osteoclast precursors. Resorption of bone substrates was prevented by a neutralising anti-RANK-L antibody. Our data indicate that osteoclast-like transformation of both U-266 and MCC-2 cellular models of human myeloma is dependent on RANK-L stimulation.

Actins↗

PubMed1989

Appearance of osteoclasts by injections of lipopolysaccharides in rat periodontal tissue.

The effect of lipopolysaccharides (LPS) on periodontal tissue was studied in 11-week-old Wistar rats. Four injections of 500 micrograms LPS induced marked osteoclastic alveolar bone resorption, whereas no alveolar bone resorption occurred after four injections of physiological saline. The osteoclast count increased progressively during the four injections of 500 micrograms LPS. After eight injections, however, the osteoclast count fell from the maximum level after four injections, although osteoclasts continued to increase in size and demonstrate an increased number of nuclei. Comparison of the osteoclast number between a series of four injections of 5, 50 or 500 micrograms LPS each revealed a dose-dependent increase. This strongly suggests LPS induction of osteoclastic bone resorption in vivo. Combined use of indomethacin, a prostaglandin synthesis inhibitor, with LPS injections inhibited both the LPS-induced alveolar bone resorption and osteoclast increase, suggesting a possible participation of prostaglandins in osteoclast-mediated bone resorption.

Animals↗

PubMed1998

Involvement of prostaglandin E2 and interleukin-1 alpha in the differentiation and survival of osteoclasts induced by lipopolysaccharide from Actinobacillus actinomycetemcomitans Y4.

Lipopolysaccharide (LPS) is a bacterial cell component that plays multifunctional roles in inflammatory reactions. LPS from various periodontal pathogens is supposed to be a major virulence factor of periodontal diseases. In the present study, we demonstrated that LPS from periodontopathic bacterium Actinobacillus actinomycetemcomitans Y4 (Y4 LPS) stimulated osteoclast formation in mouse bone marrow culture systems. Addition of anti-interleukin-1 alpha (IL-1 alpha) antibody or indomethacin in the marrow cultures resulted in the suppression of osteoclast differentiation. Quantitative analyses revealed that Y4 LPS stimulated the production of IL-1 alpha and prostaglandin E2 (PGE2) by bone marrow cells. Furthermore, an immunoblot analysis showed that Y4 LPS stimulated bone marrow cells to upregulate the expression of cyclooxygenase-2, a rate-limiting enzyme for the conversion of arachidonic acid to prostanoids. These findings suggest that both IL-1 alpha and PGE2 are involved in the LPS-mediated osteoclast differentiation. In addition, we found that Y4 LPS supported the survival of osteoclasts. Addition of anti-IL-1 alpha antibody in the osteoclast culture resulted in a reduction of osteoclast survival. Indomethacin, however, showed no effect on osteoclast survival. These findings suggest that the increased PGE2 and IL-1 alpha synthesis by bone marrow cells may play an important role in the differentiation and survival of osteoclasts induced by A. actinomycetemcomitans LPS.

Aggregatibacter actinomycetemcomitans↗

PubMed1993

Amylin inhibits bone resorption by a direct effect on the motility of rat osteoclasts.

We have performed a set of independent studies on the effects of the circulating pancreatic polypeptide, amylin, on rat osteoclast function, in vitro. Time-lapse video observations, measuring cell protrusions and retraction, showed that 250 nmol l-1 amylin or 250 nmol l-1 beta-calcitonin gene-related peptide (beta-CGRP) inhibited osteoclast motility (quiescence or Q effect). Both amylin and beta-CGRP produced inhibitory responses with a significant first-order regression over time (half-times, 19 and 28 min respectively). In contrast, 250 nmol l-1 amylin or 250 nmol l-1 beta-CGRP produced no change of osteoclast spread area, whilst 300 pmol l-1 calcitonin (CT) application resulted in cell retraction (R effect). Forskolin (10 mumol l-1) mimicked amylin and CGRP in inhibiting osteoclast motility (half-time, 8.6 min), and similarly lacked an effect on cell spread area. Neither amylin nor beta-CGRP (62.5-1250 nmol l-1) elevated cytosolic free calcium levels ([Ca2+]i) in single osteoclasts whilst 300 pmol l-1 salmon calcitonin (sCT) produced a rapid phasic elevation of [Ca2+]i, confirming previous results with asusuberic (1-7) eel calcitonin. The osteoclast-bone resorption assay revealed the following potency difference in direct comparison of the area of resorption per bone slice: beta-CGRP/amylin, 0.1; sCT/amylin, 800 and human CT/amylin, 12. The potency of deamidated amylin approached that of beta-CGRP. Assay precision ranged between 0.3 and 0.8. Amylin (250 nmol l-1) also significantly (P < 0.05) reduced supernatant (tartrate-resistant) acid phosphatase in the bone-osteoclast cultures. These measures independently indicate an effect of amylin on osteoclast motility through mechanisms distinct from those of calcitonin, possibly through different selectivities for receptor subtypes, the cyclic AMP-linked 'amylin subtype' and the [Ca2+]i-linked 'calcitonin subtype'.

Amyloid↗

PubMed1978

Effect of calcitonin treatment on osteoclast counts in Paget's disease of bone.

Histological and biochemical changes during calcitonin treatment have been studied in 15 patients with Paget's disease of bone. For each patient, osteoclast counts were made by the same observer on serial needle biopsies of diseased bone from the posterosuperior iliac spine. Serial estimations were also made of the serum alkaline phosphatase and urinary hydroxyproline excretion. A total of 66 biopsies was examined (ranging from two to seven per patient). Osteoclast populations and the biochemical measurements were log normally distributed. During calcitonin treatment there was a statistically significant decrease in: (1) the total osteoclast count per square millimetre; (2) the number per square millimetre of osteoclasts in resorption cavities on the trabecular surface; (3) the relative proportion of osteoclasts sited in resorption cavities compared with total osteoclasts; (4) the serum alkaline phosphatase level; (5) 24-hour urinary hydroxyproline excretion. On stopping treatment there was a statistically significant increase in all of these histological and biochemical values except that the proportion of osteoclasts in resorption cavities remained low. The trabecular cement line pattern remained abnormal during and after treatment in all biopsies examined, and complete suppression of osteoclast activity was not observed. One of the patients developed a Paget's osteosarcoma while on calcitonin therapy.

Aged↗

PubMed2006

Unique uptake and efflux systems of inorganic phosphate in osteoclast-like cells.

During bone resorption, a large amount of inorganic phosphate (P(i)) is generated within the osteoclast hemivacuole. The mechanisms involved in the disposal of this P(i) are not clear. In the present study, we investigated the efflux of P(i) from osteoclast-like cells. P(i) efflux was activated by acidic conditions in osteoclast-like cells derived by the treatment of RAW264.7 cells with receptor activator of nuclear factor-kappaB ligand. Acid-induced P(i) influx was not observed in renal proximal tubule-like opossum kidney cells, osteoblast-like MC3T3-E1 cells, or untreated RAW264.7 cells. Furthermore, P(i) efflux was stimulated by extracellular P(i) and several P(i) analogs [phosphonoformic acid (PFA), phosphonoacetic acid, arsenate, and pyrophosphate]. P(i) efflux was time dependent, with 50% released into the medium after 10 min. The efflux of P(i) was increased by various inhibitors that block P(i) uptake, and extracellular P(i) did not affect the transport of [(14)C]PFA into the osteoclast-like cells. Preloading of cells with P(i) did not stimulate P(i) efflux by PFA, indicating that the effect of P(i) was not due to transstimulation of P(i) transport. P(i) uptake was also enhanced under acidic conditions. Agents that prevent increases in cytosolic free Ca(2+) concentration, including acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 2-aminoethoxydiphenyl borate, and bongkrekic acid, significantly inhibited P(i) uptake in the osteoclast-like cells, suggesting that P(i) uptake is regulated by Ca(2+) signaling in the endoplasmic reticulum and mitochondria of osteoclast-like cells. These results suggest that osteoclast-like cells have a unique P(i) uptake/efflux system and can prevent P(i) accumulation within osteoclast hemivacuoles.

Animals↗

PubMed2002

Cytokines synergistically induce osteoclast differentiation: support by immortalized or normal calvarial cells.

Conditionally immortalized murine calvarial (CIMC) cells that support differentiation of precursors into mature osteoclasts were isolated. All six CIMC cell lines supported osteoclast differentiation in response to 1,25-dihydroxyvitamin D(3) or interleukin (IL)-11. CIMC-4 cells also supported osteoclast differentiation in response to tumor necrosis factor (TNF)-alpha, IL-1beta, or IL-6. The resultant multinucleated cells expressed tartrate-resistant acid phosphatase and formed resorption lacunae on mineralized surfaces. CIMC-4 cells, therefore, establish an osteoclast differentiation assay that is responsive to many cytokines and does not rely on isolation of primary stromal support cells. Low concentrations of the cytokines synergistically stimulated differentiation when osteoclast precursors were cocultured with either CIMC-4 cells or primary calvarial cells. Osteoclast differentiation induced by all stimuli other than TNF-alpha was completely blocked by osteoprotegerin, whether the stimulators were examined alone or in combination. Moreover, study of precursors that lack TNF-alpha receptors showed that TNF-alpha induces osteoclast differentiation primarily through direct actions on osteoclast precursors, which is a distinct mechanism from that used by the other bone-resorptive agents examined in this study.

Acid Phosphatase↗

PubMed1994

Role of interleukin-6 and prostaglandins in the effect of monocyte-conditioned medium on osteoclast formation.

There is evidence suggesting some role of mononuclear cells at the resorptive site in bone remodeling. The possibility was therefore postulated that these cells might provide some signal for osteoclast formation. We examined the effects of human monocyte-conditioned medium (CM) on the formation of osteoclast-like cells from hemopoietic blast cells in the absence of stromal cells and unfractionated bone cells in the presence of stromal cells. In both culture systems, the osteoclast-like cell formation induced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or human parathyroid hormone (PTH)-(1-34) was significantly inhibited by adding 20% CM. The effects of monocyte-derived local regulators of bone turnover on osteoclast-like cell formation induced by 1,25(OH)2D3 or PTH were determined. Interleukin-6 (IL-6) inhibited osteoclast-like cell formation in both culture systems, whereas prostaglandin (PG) E2 significantly inhibited the formation only in the hemopoietic blast cell culture. The inhibitory effect of CM on osteoclast-like cell formation from hemopoietic blast cells was not observed when CM was prepared from monocytes pretreated with indomethacin. The inhibitory effect of CM and IL-6 on osteoclast-like cell formation in both culture systems was blocked by adding neutralizing IL-6 antibody. The present study demonstrated that CM inhibited osteoclast-like cell formation induced by 1,25(OH)2D3 and PTH presumably through the action of local regulators of bone turnover, such as IL-6 and PG. Our findings thus provide additional evidence that mononuclear cells play an important role at the resorptive site in bone remodeling.

Acid Phosphatase↗

PubMed1995

Local concentrations of macrophage colony-stimulating factor mediate osteoclastic differentiation.

Macrophage colony-stimulating factor (M-CSF) is essential for differentiation of osteoclasts and macrophages from a common bone marrow precursor. Using ST-2 stromal cell/murine bone marrow coculture, we studied the effects of increasing amounts of M-CSF on differentiation of macrophages and osteoclasts. Addition of exogenous M-CSF caused a dose-dependent 98% decrease in tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, accompanied by a 2.5-fold increase in nonspecific esterase-staining macrophages. Similar decrease in osteoclastic functional activity, including 125I-labeled calcitonin binding and calcitonin-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production, were observed. Addition of exogenous M-CSF beyond 6 days in coculture had a decreasing ability to inhibit osteoclast formation, suggesting that M-CSF exerts its effects early in osteoclast differentiation, during the proposed proliferative phase of osteoclast formation. Similarly, early addition of neutralizing anti-M-CSF inhibited osteoclast formation, with diminishing effects beyond day 9. These results suggest that local high concentrations of M-CSF may influence the early determination of terminal differentiation into either macrophages or osteoclasts.

Acid Phosphatase↗

PubMed2001

pH dependence of bone resorption: mouse calvarial osteoclasts are activated by acidosis.

We examined the effects of HCO(3)(-) and CO(2) acidosis on osteoclast-mediated Ca(2+) release from 3-day cultures of neonatal mouse calvaria. Ca(2+) release was minimal above pH 7.2 in control cultures but was stimulated strongly by the addition of small amounts of H(+) to culture medium (HCO(3)(-) acidosis). For example, addition of 4 meq/l H(+) reduced pH from 7.12 to 7.03 and increased Ca(2+) release 3.8-fold. The largest stimulatory effects (8- to 11-fold), observed with 15-16 meq/l added H(+), were comparable to the maximal Ca(2+) release elicited by 1,25-dihydroxyvitamin D(3) [1, 25(OH)(2)D(3); 10 nM], parathyroid hormone (10 nM), or prostaglandin E(2) (1 microM); the action of these osteolytic agents was attenuated strongly when ambient pH was increased from approximately 7.1 to approximately 7.3. CO(2) acidosis was a less effective stimulator of Ca(2+) release than HCO(3)(-) acidosis over a similar pH range. Ca(2+) release stimulated by HCO(3)(-) acidosis was almost completely blocked by salmon calcitonin (20 ng/ml), implying osteoclast involvement. In whole mount preparations of control half-calvaria, approximately 400 inactive osteoclast-like multinucleate cells were present; in calvaria exposed to HCO(3)(-) acidosis and to the other osteolytic agents studied, extensive osteoclastic resorption, with perforation of bones, was visible. HCO(3)(-) acidosis, however, reduced numbers of osteoclast-like cells by approximately 50%, whereas 1,25(OH)(2)D(3) treatment caused increases of approximately 75%. The results suggest that HCO(3)(-) acidosis stimulates resorption by activating mature osteoclasts already present in calvarial bones, rather than by inducing formation of new osteoclasts, and provide further support for the critical role of acid-base balance in controlling osteoclast function.

Acid-Base Equilibrium↗

PubMed2002

Molecular cloning, expression, and function of osteoclastic calcineurin Aalpha.

This study explores the role of the calmodulin- and Ca(2+)-sensitive phosphatase calcineurin A in the control of bone resorption by mature osteoclasts. We first cloned full-length calcineurin Aalpha and Abeta cDNA from a rabbit osteoclast library. Sequence analysis revealed an approximately 95 and 86% homology between the amino acid and the nucleotide sequences, respectively, of the two isoforms. The two rabbit isoforms also showed significant homology with the mouse, rat, and human homologs. In situ RT-PCR showed evidence of high levels of expression of calcineurin Aalpha mRNA in freshly isolated rat osteoclasts. Semiquantitative analysis of staining intensity revealed no significant difference in calcineurin Aalpha expression in cells treated with vehicle vs. those treated with the calcineurin (activity) inhibitors cyclosporin A (8 x 10(-7) M) and FK506 (5 x 10(-9) and 5 x 10(-7) M). We then constructed a fusion protein comprising calcineurin Aalpha and TAT, a 12-amino acid-long arginine-rich sequence of the human immunodeficiency virus protein. Others have previously shown that the fusion of proteins to this sequence results in their receptor-less transduction into cells, including osteoclasts. Similarly, unfolding of the TAT-calcineurin Aalpha fusion protein by shocking with 8 M urea resulted in its rapid influx, within minutes, into as many as 90% of all freshly isolated rat osteoclasts, as was evident on double immunostaining with anti-calcineurin Aalpha and anti-TAT antibodies. Pit assays performed with TAT-calcineurin Aalpha-positive osteoclasts revealed a concentration-dependent (10-200 nM) attenuation of bone resorption in the absence of cell cytotoxicity or changes in cell number. TAT-hemaglutinin did not produce significant effects on bone resorption or cell number. The study suggests the following: 1) the 61-kDa protein phosphatase calcineurin Aalpha can be effectively tranduced into osteoclasts by using the TAT-based approach, and 2) the transduced protein retains its capacity to inhibit osteoclastic bone resorption.

Amino Acid Sequence↗

PubMed2006

Evidence that calcineurin is required for the genesis of bone-resorbing osteoclasts.

Here, we demonstrate that the Ca(2+)/calmodulin-sensitive phosphatase calcineurin is a necessary downstream mediator for osteoclast differentiation. Using quantitative PCR, we detected the calcineurin isoforms Aalpha, Abeta, Agamma (catalytic), and B1 (regulatory) in osteoclast precursor RAW-C3 cells. We found that, although the expression of these isoforms remained relatively unchanged during osteoclast differentiation, there was a profound increase in the expression of their primary substrate for calcineurin, nuclear factor of activated T cells (NFAT)c1. For gain-of-function studies, we incubated osteoclast precursors for 10 min with a calcineurin fusion protein (TAT-calcineurin Aalpha); this resulted in its receptorless influx into >90% of the precursor cells. A marked increase in the expression of the osteoclast differentiation markers tartrate-resistant acid phosphatase (TRAP) and integrin beta(3) followed. In addition, the expression of NFATc1, as well as the alternative substrate for calcineurin, IkappaBalpha, was significantly enhanced. Likewise, transfection with constitutively active NFAT resulted in an increased expression of both TRAP and integrin beta(3). In parallel loss-of-function studies, transfection with dominant-negative NFAT not only inhibited osteoclast formation but also reversed the induction of NFATc1, TRAP, and integrin beta(3) by TAT-calcineurin Aalpha. The expression of these markers was also inhibited by calcineurin Aalpha U1 small nuclear RNA, which significantly reduced calcineurin Aalpha mRNA and protein expression. Consistent with these observations, we observed a reduction in osteoclastogenesis in calcineurin Aalpha(-/-) cells and in osteoclast precursors treated with the calcineurin inhibitors cyclosporin A and FK506. Together, the gain- and loss-of-function experiments establish that calcineurin Aalpha is necessary for osteoclast formation from its precursor and that this occurs via an NFATc1-dependent mechanism.

Acid Phosphatase↗

PubMed2006

A c-fms tyrosine kinase inhibitor, Ki20227, suppresses osteoclast differentiation and osteolytic bone destruction in a bone metastasis model.

In bone metastatic lesions, osteoclasts play a key role in the development of osteolysis. Previous studies have shown that macrophage colony-stimulating factor (M-CSF) is important for the differentiation of osteoclasts. In this study, we investigated whether an inhibitor of M-CSF receptor (c-Fms) suppresses osteoclast-dependent osteolysis in bone metastatic lesions. We developed small molecule inhibitors against ligand-dependent phosphorylation of c-Fms and examined the effects of these compounds on osteolytic bone destruction in a bone metastasis model. We discovered a novel quinoline-urea derivative, Ki20227 (N-{4-[(6,7-dimethoxy-4-quinolyl)oxy]-2-methoxyphenyl}-N'-[1-(1,3-thiazole-2-yl)ethyl]urea), which is a c-Fms tyrosine kinase inhibitor. The IC(50)s of Ki20227 to inhibit c-Fms, vascular endothelial growth factor receptor-2 (KDR), stem cell factor receptor (c-Kit), and platelet-derived growth factor receptor beta were found to be 2, 12, 451, and 217 nmol/L, respectively. Ki20227 did not inhibit other kinases tested, such as fms-like tyrosine kinase-3, epidermal growth factor receptor, or c-Src (c-src proto-oncogene product). Ki20227 was also found to inhibit the M-CSF-dependent growth of M-NFS-60 cells but not the M-CSF-independent growth of A375 human melanoma cells in vitro. Furthermore, in an osteoclast-like cell formation assay using mouse bone marrow cells, Ki20227 inhibited the development of tartrate-resistant acid phosphatase-positive osteoclast-like cells in a dose-dependent manner. In in vivo studies, oral administration of Ki20227 suppressed osteoclast-like cell accumulation and bone resorption induced by metastatic tumor cells in nude rats following intracardiac injection of A375 cells. Moreover, Ki20227 decreased the number of tartrate-resistant acid phosphatase-positive osteoclast-like cells on bone surfaces in ovariectomized (ovx) rats. These findings suggest that Ki20227 inhibits osteolytic bone destruction through the suppression of M-CSF-induced osteoclast accumulation in vivo. Therefore, Ki20227 may be a useful therapeutic agent for osteolytic disease associated with bone metastasis and other bone diseases.

Acid Phosphatase↗

PubMed1999

Calcium signalling via multiple P2 purinoceptor subtypes in rat osteoclasts.

Extracellular nucleotides bind to P2 purinoceptors in many tissues. P2X purinoceptors are intrinsic ion channels that mediate depolarization and influx of Ca(2+), whereas P2Y purinoceptors are coupled through G-proteins to mobilization of intracellular Ca(2+). Previous studies have yielded conflicting information on the responses of osteoclasts to nucleotides. The purpose of this study was to investigate the pathways underlying purinoceptor-mediated Ca(2+) signalling in authentic mammalian osteoclasts. Osteoclasts, isolated from the long bones of neonatal rats, were loaded with the Ca(2+)-sensitive probe fura-2 and [Ca(2+)](i) was monitored by microspectrofluorimetry. ATP (10-100 microM) induced transient elevation of [Ca(2+)](i) in 74% of osteoclasts tested. Similar responses were observed in Ca(2+)-free media, consistent with release of Ca(2+) from intracellular stores. Oscillations in [Ca(2+)](i) were observed only in osteoclasts that had a 'rounded' morphology. Responses to selective P2 agonists were consistent with the presence of multiple purinoceptor subtypes, including members of both the P2Y and P2X families. Alendronate, a bisphosphonate with structural similarities to methylene ATP analogues, neither activated nor blocked the Ca(2+) response mediated by osteoclast purinoceptors. Mechanical stimulation of osteoclasts elicited transient elevation of [Ca(2+)](i) which involved Ca(2+) influx and, in some cases, release from stores. The nucleotidase apyrase did not inhibit deformation-induced elevation of [Ca(2+)](i) in the presence of extracellular Ca(2+), indicating that nucleotide release is not essential for mechanically induced Ca(2+) influx. These findings indicate that osteoclasts exhibit multiple P2 purinoceptor subtypes, linked to elevation of [Ca(2+)](i).

Adenosine Triphosphate↗