Serum calcium-decreasing factor, caldecrin, inhibits receptor activator of NF-κB ligand (RANKL)-mediated Ca2+ signaling and actin ring formation in mature osteoclasts via suppression of Src signaling pathway

Osteoclasts are essential for bone dynamics and calcium homeostasis. Recently, we reported that serum calcium-decreasing factor, caldecrin, which is a secretory-type serine protease isolated from the pancreas, inhibits osteoclast differentiation by suppression of NFATc1 activity regardless of its own protease activity (Hasegawa, H., Kido, S., Tomomura, M., Fujimoto, K., Ohi, M., Kiyomura, M., Kanegae, H., Inaba, A., Sakagami, H., and Tomomura, A. (2010) Serum calcium-decreasing factor, caldecrin, inhibits osteoclast differentiation by suppression of NFATc1 activity. J. Biol. Chem. 285, 25448-25457). Here, we investigated the effects of caldecrin on the function of mature osteoclasts by treatment with receptor activator of NF-κB ligand (RANKL). Caldecrin inhibited the RANKL-stimulated bone resorptive activity of mature osteoclasts. Furthermore, caldecrin inhibited RANKL-mediated sealing actin ring formation, which is associated with RANKL-evoked Ca(2+) entry through transient receptor potential vanilloid channel 4. The inhibitors of phospholipase Cγ, Syk, and c-Src suppressed RANKL-evoked Ca(2+) entry and actin ring formation of mature osteoclasts. Interestingly, caldecrin significantly inhibited RANKL-stimulated phosphorylation of c-Src, Syk, phospholipase Cγ1 and Cγ2, SLP-76, and Pyk2 but not that of ERK, JNK, or Akt. Caldecrin inhibited RANKL-stimulated c-Src kinase activity and c-Src·Syk association. These results suggest that caldecrin inhibits RANKL-stimulated calcium signaling activation and cytoskeletal organization by suppression of the c-Src·Syk pathway, which may in turn reduce the bone resorptive activity of mature osteoclasts. Thus, caldecrin is capable of acting as a negative regulator of osteoclastogenesis and osteoclast function of bone resorption.     

Protein tyrosine kinase inhibitors increase cytosolic calcium and inhibit actin organization as resorbing activity in rat osteoclasts

Although there is evidence that protein tyrosine kinase inhibitors (PTKIs) suppress bone resorption activity, the mechanism of action of these compounds on osteoclastic bone resorption remains obscure. In the present study, we investigated the effect of PTKIs on cytosolic Ca(2+) concentration ([Ca(2+)](i)) and on the cytoskeleton in rat osteoclasts. The PTKIs, genistein and herbimycin A, reversibly elevated [Ca(2+)](i) measured by fura-2 microfluorimetry. The PTKI-induced increase was abolished by omission of extracellular Ca(2+), but was not attenuated by depletion of Ca(2+) stores. The PTKI-induced increase was inhibited by addition of La(3+) and Ni(2+), but not abolished by dihydropyridine (DHP) Ca(2+) channel blockers. Genistin, an inactive analogue of genistein, had no effect on [Ca(2+)](i). In the cytoskeleton assay, genistein rapidly disrupted the actin ring formation that serves as a marker for the resorbing state of osteoclasts. Disruption of the actin ring formation was also diminished in Ca(2+)-free extracellular solution. These results suggest that PTKIs in rat osteoclasts elevate [Ca(2+)](i) via activation of a DHP-insensitive, nonspecific Ca(2+) entry pathway and disrupt the formation of actin rings, resulting in suppression of bone resorption activity. The regulation of this Ca(2+)-influx by PTKIs is likely to contribute to inhibition of bone resorption by these compounds.     

Evidence of reciprocal regulation between the high extracellular calcium and RANKL signal transduction pathways in RAW cell derived osteoclasts

During bone resorption, osteoclasts are exposed to high Ca2+ concentrations (up to 40 mM). The role of high extracellular Ca2+ in receptor activator of NF-kappaB ligand (RANKL)-mediated osteoclast survival and their functional interrelationship is unclear. In this study, we show that RANKL enhances osteoclast tolerance to high extracellular Ca2+ by protecting the cell from cell death in a dose dependent manner. We have provided evidence that RANKL does this by attenuating high extracellular Ca2+-induced Ca2+ elevations. Moreover, we have found that high extracellular Ca2+-induced cell death was partially inhibited by a caspase-3 inhibitor, suggesting caspase-3-mediated apoptosis is involved. Conversely, using reporter gene assays and Western blot analysis, we have demonstrated that high extracellular Ca2+ desensitizes the RANKL-induced activation of NF-kappaB and c-Jun N-terminal kinase (JNK), and inhibits constitutive and RANKL-stimulated ERK phosphorylation, indicating a negative feed-back mechanism via specific RANKL signaling pathways. Taken together, this study provides evidence for a reciprocal regulation between high extracellular Ca2+ and RANKL signaling in RAW cell derived osteoclasts. Our data imply a cross talk mechanism of extracellular Ca2+ on osteoclast survival through the regulation of RANKL.     

Effect of high phosphate concentration on osteoclast differentiation as well as bone-resorbing activity

Although high inorganic phosphate (Pi) concentration in culture media directly inhibits generation of new osteoclasts and also inhibits bone resorption by mature osteoclasts, its precise mechanism and the physiological role have not been elucidated. The present study was performed to investigate these issues. Increase in extracellular Pi concentration ([Pi](e)) (2.5-4 mM) concentration dependently inhibited 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] or parathyroid hormone (PTH)-(1-34)-induced osteoclast-like cell formation from unfractionated bone cells in the presence of stromal cells. Increase in [Pi](e) (2.5-4 mM) concentration dependently inhibited 1,25(OH)(2)D(3)-, PTH-(1-34)-, or receptor activator of NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF)-induced osteoclast-like cell formation from hemopoietic blast cells in the absence of stromal cells. Increase in [Pi](e) (2.5-4 mM) dose dependently stimulated the expression of osteoprotegerin (OPG) mRNA and increased the expression of OPG mRNA suppressed by PTH-(1-34) or 1,25(OH)(2)D(3) in unfractionated bone cells, while it did not affect RANKL mRNA. Increase in [Pi](e) (2.5-4 mM) concentration dependently inhibited the bone-resorbing activity of isolated rabbit osteoclasts. Increase in [Pi](e) (4 mM) induced the apoptosis of isolated rabbit osteoclasts while it did not affect the apoptosis of osteoclast precursor cells and mouse macrophage-like cell line C7 cells that can differentiate into osteoclasts in the presence of RANKL and M-CSF. These results indicate that increase in [Pi](e) inhibits osteoclast differentiation both by up-regulating OPG expression and by direct action on osteoclast precursor cells. It is also indicated that increase in [Pi](e) inhibits osteoclastic activity at least in part by the direct induction of apoptosis of osteoclasts.     

Atp6v1c1 is an essential component of the osteoclast proton pump and in F-actin ring formation in osteoclasts

Bone resorption relies on the extracellular acidification function of V-ATPase (vacuolar-type proton-translocating ATPase) proton pump(s) present in the plasma membrane of osteoclasts. The exact configuration of the osteoclast-specific ruffled border V-ATPases remains largely unknown. In the present study, we found that the V-ATPase subunit Atp6v1c1 (C1) is highly expressed in osteoclasts, whereas subunits Atp6v1c2a (C2a) and Atp6v1c2b (C2b) are not. The expression level of C1 is highly induced by RANKL [receptor activator for NF-kappaB (nuclear factor kappaB) ligand] during osteoclast differentiation; C1 interacts with Atp6v0a3 (a3) and is mainly localized on the ruffled border of activated osteoclasts. The results of the present study show for the first time that C1-silencing by lentivirus-mediated RNA interference severely impaired osteoclast acidification activity and bone resorption, whereas cell differentiation did not appear to be affected, which is similar to a3 silencing. The F-actin (filamentous actin) ring formation was severely defected in C1-depleted osteoclasts but not in a3-depleted and a3(-/-) osteoclasts. C1 co-localized with microtubules in the plasma membrane and its vicinity in mature osteoclasts. In addition, C1 co-localized with F-actin in the cytoplasm; however, the co-localization chiefly shifted to the cell periphery of mature osteoclasts. The present study demonstrates that Atp6v1c1 is an essential component of the osteoclast proton pump at the osteoclast ruffled border and that it may regulate F-actin ring formation in osteoclast activation.     

Regulation of Sealing Ring Formation by L-plastin and Cortactin in Osteoclasts

The aim of this study is to identify the exact mechanism(s) by which cytoskeletal structures are modulated during bone resorption. In this study, we have shown the possible role of different actin-binding and signaling proteins in the regulation of sealing ring formation. Our analyses have demonstrated a significant increase in cortactin and a corresponding decrease in L-plastin protein levels in osteoclasts subjected to bone resorption for 18 h in the presence of RANKL, M-CSF, and native bone particles. Time-dependent changes in the localization of L-plastin (in actin aggregates) and cortactin (in the sealing ring) suggest that these proteins may be involved in the initial and maturation phases of sealing ring formation, respectively. siRNA to cortactin inhibits this maturation process but not the formation of actin aggregates. Osteoclasts treated as above but with TNF-α demonstrated very similar effects as observed with RANKL. Osteoclasts treated with a neutralizing antibody to TNF-α displayed podosome-like structures in the entire subsurface and at the periphery of osteoclast. It is possible that TNF-α and RANKL-mediated signaling may play a role in the early phase of sealing ring configuration (i.e. either in the disassembly of podosomes or formation of actin aggregates). Furthermore, osteoclasts treated with alendronate or αv reduced the formation of the sealing ring but not actin aggregates. The present study demonstrates a novel mechanistic link between L-plastin and cortactin in sealing ring formation. These results suggest that actin aggregates formed by L-plastin independent of integrin signaling function as a core in assembling signaling molecules (integrin αvβ3, Src, cortactin, etc.) involved in the maturation process.     

Caldecrin:A pancreas-derived hypocalcemic factor,regulates osteoclast formation and function

Caldecrin was originally isolated from the pancreas as afactor that reduced serum calcium levels. This secreted serine protease has chymotrypsin-like activity and is also known as chymotrypsin C; it belongs to the elastase family. Although intravenous administration of caldecrin decreases the serum calcium concentration even when its protease activity is blocked,this effect does require cleavage of caldecrin's pro-peptide by trypsin,converting it to the mature enzyme. Ectopic intramuscular expression of caldecrin prevented bone resorption in ovariectomized mice. Caldecrin inhibited parathyroid hormone-stimulated calcium release from fetal mouse long bone organ cultures. Furthermore,caldecrin suppressed the formation of osteoclasts from bone marrow cells by inhibiting the receptor activator of nuclear factor-k B ligand(RANKL)-stimulated phospholipase Cγ-calcium oscillation-calcineurinnuclear factor of activated T-cells,cytoplasmic 1 pathway. Caldecrin also suppressed the bone resorption activity of mature osteoclasts by preventing RANKL-stimulated Src activation,calcium entry,and actin ring formation. In vivo and in vitro studies have indicated that caldecrin is a unique multifunctional protease with anti-osteoclastogenic activities that are distinct from its protease activity. Caldecrin might be a potential therapeutic target for the treatment of osteolytic diseases such as osteoporosis and osteoarthritis. This mini-review describes caldecrin's historical background and its mechanisms of action.     

miR-31 controls osteoclast formation and bone resorption by targeting RhoA

Introduction

Increased activity of osteoclasts is responsible for bone loss and joint destruction in rheumatoid arthritis. For osteoclast development and bone resorption activity, cytoskeletal organization must be properly regulated. MicroRNAs (miRNAs) are endogenous small noncoding RNAs that suppress expression of their target genes. This study was conducted to identify crucial miRNAs to control osteoclasts.

Methods

miRNA expression in the bone marrow-derived macrophages (BMM) with or without receptor activator of nuclear factor κB ligand (RANKL) stimulation was analyzed by miRNA array. To examine the role of specific miRNAs in osteoclast formation, bone resorption activity and actin ring formation, the BMM were retrovirally transduced with miRNA antagomirs. To confirm whether the suppressive effects on osteoclastogenesis by miR-31 inhibition were mediated by targeting RhoA, osteoclast formation was analyzed in the presence of the RhoA inhibitor, exoenzyme C3.

Results

miR-31 was identified as one of the highly upregulated miRNAs during osteoclast development under RANKL stimulation. Inhibition of miR-31 by specific antagomirs suppressed the RANKL-induced formation of osteoclasts and bone resorption. Phalloidin staining of osteoclasts revealed that actin ring formation at the cell periphery was severely impaired by miR-31 inhibition, and clusters of small ringed podosomes were observed instead. In these osteoclasts, expression of RhoA, one of the miR-31 target genes, was upregulated by miR-31 inhibition in spite of the impaired osteoclastogenesis. Treatment with the RhoA inhibitor, exoenzyme C3, rescued the osteoclastogenesis impaired by miR-31 inhibition.

Conclusions

miR-31 controls cytoskeleton organization in osteoclasts for optimal bone resorption activity by regulating the expression of RhoA.     

Inhibitory effect of luteolin on osteoclast differentiation and function

Osteoclasts are multinucleated cells that play a crucial role in bone resorption, and are formed by the fusion of mononuclear osteoclasts derived from osteoclast precursors of the macrophage lineage. Compounds that specifically target functional osteoclasts would be ideal candidates for anti-resorptive agents for clinical applications. In the present study, we investigated the effects of luteolin, a flavonoid, on the regulation of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, functions and signaling pathway. Addition of luteolin to a coculture system of mouse bone marrow cells and ST2 cells in the presence of 10⁻⁸ M 1α,25(OH)₂D₃ caused significant inhibition of osteoclastogenesis. Luteolin had no effects on the 1α,25(OH)₂D₃-induced expressions of RANKL, osteoprotegerin and macrophage colony-stimulating factor mRNAs. Next, we examined the direct effects of luteolin on osteoclast precursors using bone marrow macrophages and RAW264.7 cells. Luteolin completely inhibited RANKL-induced osteoclast formation. Moreover, luteolin inhibited the bone resorption by mature osteoclasts accompanied by the disruption of their actin rings, and these effects were reversely induced by the disruption of the actin rings in mature osteoclasts. Finally, we found that luteolin inhibited RANKL-induced osteoclastogenesis through the suppression of ATF2, downstream of p38 MAPK and nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) expression, respectively. Taken together, the present results indicate that naturally occurring luteolin has inhibitory activities toward both osteoclast differentiation and functions through inhibition of RANKL-induced signaling pathway as well as actin ring disruption, respectively.     

Fisetin antagonizes cell fusion,cytoskeletal organization and bone resorption in RANKL-differentiated murine macrophages

Osteoclastogenesis is comprised of several stage s including progenitor survival, differentiation to mononuclear preosteoclasts, cell fusion to multinuclear mature osteoclasts, and activation to osteoclasts with bone resorbing activity. Botanical antioxidants are now being increasingly investigated for their health-promoting effects on bone. This study investigated that fisetin, a flavonol found naturally in many fruits and vegetables, suppressed osteoclastogenesis by disturbing receptor activator of nuclear factor (NF)-κB ligand (RANKL)-mediated signaling pathway and demoting osteoclastogenic protein induction. Nontoxic fisetin at ≤10μM inhibited the induction of RANK, tumor necrosis factor receptor associated factor 6 (TRAF6) and the activation of NF-κB in RANKL-stimulated RAW 264.7 macrophages. In RANKL-differentiated osteoclasts cell fusion protein of E-cadherin was induced, which was dampened by fisetin. The formation of tartrate-resistance acid phosphatase-positive multinucleated osteoclasts was suppressed by adding fisetin to RANKL-exposed macrophages. It was also found that fisetin reduced actin ring formation and gelsolin induction of osteclasts enhanced by RANKL through disturbing c-Src-proline-rich tyrosine kinase 2 signaling. Fisetin deterred preosteoclasts from the cell-cell fusion and the organization of the cytoskeleton to seal the resorbing area and to secret protons for bone resorption. Consistently, the 5 day-treatment of fisetin diminished RANKL-induced cellular expression of carbonic anhydrase II and integrin β3 concurrently with a reduction of osteoclast bone-resorbing activity. Therefore, fisetin was a natural therapeutic agent retarding osteoclast fusion and cytoskeletal organization such as actin rings and ruffled boarder, which is a property of mature osteoclasts and is required for osteoclasts to resorb bone.     

Diosmetin inhibits osteoclast formation and differentiation and prevents LPS-induced osteolysis in mice

Osteolytic bone diseases are closely linked to the over-activation of osteoclasts and enhancement of bone resorption. It has become a major health issue in orthopedic practice worldwide. Inhibition of osteoclasts is proposed to be the main treatment for osteolytic disorders. Diosmetin (DIO) is a natural flavonoid with properties of antioxidant, anti-infection, and antishock. The effect of DIO on osteoclast differentiation is poorly understood. In this study project, we found that DIO could inhibit osteoclastic formation induced by receptor activator of nuclear factor kappa-B ligand (RANKL) in a dose-dependent manner. The expression of the osteoclast differentiation marker genes, cathepsin K, nuclear factor of activated T-cells 1 (NFATc1), Acp5, Ctr, Atp6v0d2, and Mmp9 were also decreased by the treatment of DIO. In addition, DIO attenuated the formation of actin ring and the ability of bone resorption. Further, the western blotting showed that DIO inhibits the phosphorylation of the mitogen-activated protein kinases signaling pathway induced by RANKL, accompanied by the downregulation of NFATc1 and c-Fos expression. We also found that DIO could reduce the accumulation of reactive oxygen species (ROS) induced by RANKL. In vivo, the study revealed that DIO can significantly reduce LPS-induced osteolysis in mice. Collectively, our study shows that DIO can inhibit osteoclast formation and activation, and could serve as a potential therapeutic drug for osteolytic bone diseases.     

IL-6, leukemia inhibitory factor,and oncostatin M stimulate bone resorption and regulate the expression of receptor activator of NF-kappa B ligand,osteoprotegerin, and receptor activator of NF-kappa B in mouse calvariae

IL-6, leukemia inhibitory factor (LIF), and oncostatin M (OSM) are IL-6-type cytokines that stimulate osteoclast formation and function. In the present study, the resorptive effects of these agents and their regulation of receptor activator of NF-kappaB ligand (RANKL), RANK, and osteoprotegerin (OPG) were studied in neonatal mouse calvaria. When tested separately, neither human (h) IL-6 nor the human soluble IL-6R (shIL-6R) stimulated bone resorption, but when hIL-6 and the shIL-6R were combined, significant stimulation of both mineral and matrix release from bone explants was noted. Semiquantitative RT-PCR showed that hIL-6 plus shIL-6R enhanced the expression of RANKL and OPG in calvarial bones, but decreased RANK expression. Human LIF, hOSM, and mouse OSM (mOSM) also stimulated 45Ca release and enhanced the mRNA expression of RANKL and OPG in mouse calvaria, but had no effect on the expression of RANK. In agreement with the RT-PCR analyses, ELISA measurements showed that both hIL-6 plus shIL-6R and mOSM increased RANKL and OPG proteins. 1,25-Dihydroxyvitamin D3 (D3) also increased the RANKL protein level, but decreased the protein level of OPG. OPG inhibited 45Ca release stimulated by RANKL, hIL-6 plus shIL-6R, hLIF, hOSM, mOSM, and D3. An Ab neutralizing mouse gp130 inhibited 45Ca release induced by hIL-6 plus shIL-6R. These experiments demonstrated stimulation of calvarial bone resorption and regulation of mRNA and protein expression of RANKL and OPG by D3 and IL-6 family cytokines as well as regulation of RANK expression in preosteoclasts/osteoclasts of mouse calvaria by D3 and hIL-6 plus shIL-6R.     

Clathrin-dependent endocytosis of membrane-bound RANKL in differentiated osteoclasts

Bone is continuously repaired and remodelled through well-coordinated activity of osteoblasts that form new bone and osteoclasts, which resorb it. Osteoblasts synthesize and secrete two key molecules that are important for osteoclast differentiation, namely the ligand for the receptor of activator of nuclear factor κB (RANKL) and its decoy receptor osteoprotegerin (OPG). Active membrane transport is a typical feature of the resorbing osteoclast during bone resorption. Normally, one resorption cycle takes several hours as observed by monitoring actin ring formation and consequent disappearance in vitro. During these cyclic changes, the cytoskeleton undergoes remarkable dynamic rearrangement. Active cells show a continuous process of exocytosis that plays an essential role in transport of membrane components, soluble molecules and receptor-mediated ligands thus allowing them to communicate with the environment. The processes that govern intracellular transport and trafficking in mature osteoclasts are poorly known. The principal methodological problem that have made these studies difficult is a physiological culture of osteoclasts that permit observing the vesicle apparatus in conditions similar to the in vivo conditions. In the present study we have used a number of morphological approaches to characterize the composition, formation and the endocytic and biosynthetic pathways that play roles in dynamics of differentiation of mature bone resorbing cells using a tri-dimensional system of physiologic coculture.Key words: osteoclast, podosomes, ruffled border, intracellular signal, clathrin, endocytosis, receptor activator of NF-kB ligand (RANKL).     

The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts

Osteoprotegerin (OPG) and OPG-ligand (OPGL) potently inhibit and stimulate, respectively, osteoclast differentiation (Simonet, W.S., D.L. Lacey, C.R. Dunstan, M. Kelley, M.-S. Chang, R. Luethy, H.Q. Nguyen, S. Wooden, L. Bennett, T. Boone, et al. 1997. Cell. 89:309-319; Lacey, D.L., E. Timms, H.-L. Tan, M.J. Kelley, C.R. Dunstan, T. Burgess, R. Elliott, A. Colombero, G. Elliott, S. Scully, et al. 1998. Cell. 93: 165-176), but their effects on mature osteoclasts are not well understood. Using primary cultures of rat osteoclasts on bone slices, we find that OPGL causes approximately sevenfold increase in total bone surface erosion. By scanning electron microscopy, OPGL-treated osteoclasts generate more clusters of lacunae on bone suggesting that multiple, spatially associated cycles of resorption have occurred. However, the size of individual resorption events are unchanged by OPGL treatment. Mechanistically, OPGL binds specifically to mature OCs and rapidly (within 30 min) induces actin ring formation; a marked cytoskeletal rearrangement that necessarily precedes bone resorption. Furthermore, we show that antibodies raised against the OPGL receptor, RANK, also induce actin ring formation. OPGL-treated mice exhibit increases in blood ionized Ca++ within 1 h after injections, consistent with immediate OC activation in vivo. Finally, we find that OPG blocks OPGL's effects on both actin ring formation and bone resorption. Together, these findings indicate that, in addition to their effects on OC precursors, OPGL and OPG have profound and direct effects on mature OCs and indicate that the OC receptor, RANK, mediates OPGL's effects.     

The glutamate receptor antagonist MK801 modulates bone resorption in vitro by a mechanism predominantly involving osteoclast differentiation.

Recent identification in bone of transporters, receptors, and components of synaptic signaling suggests a role for glutamate in the skeleton. We investigated effects of glutamate and its antagonist MK801 on osteoclasts in vitro. Glutamate applied to patch clamped osteoclasts induced significant increases in whole-cell membrane currents (P<0.01) in the presence of the coagonist glycine. Agonist-elicited currents were significantly decreased after application of MK801 (100 microM, P<0.01), but MK801 had no effect on actin ring formation necessary for osteoclast polarization, attachment, and resorption. In cocultures of bone marrow cells and osteoblasts in which osteoclasts develop, MK801 inhibited osteoclast differentiation and reduced resorption of pits in dentine (3 to 100 microM; P<0.001). MK801 added early in the culture (for as little as 2-4 days) was as effective as addition for the entire culture period. Addition of MK801 for any time after day 7 of culture was ineffective in reducing osteoclast activity. Using rat and rabbit mature osteoclasts cultured on dentine or explants of mouse calvariae prelabeled with (45)Ca, we could not detect significant effects of MK801 on osteoclastic resorption. These data show clearly that glutamate receptor function is critical during osteoclastogenesis and suggest that glutamate is less important in regulating mature osteoclast activity.-Peet, N. M., Grabowski, P. S., Laketic-Ljubojevic, I., Skerry, T. M. The glutamate receptor antagonist MK801 modulates bone resorption in vitro by a mechanism predominantly involving osteoclast differentiation.     

Microscopic study on resorption of β-tricalcium phosphate materials by osteoclasts

Sintered compounds prepared with β-tricalcium phosphate (β-TCP) are commonly used as biocompatible materials for bone regenerative medicine. Although implanted β-TCP is gradually replaced with new bone after resorption by osteoclasts, exactly how osteoclasts resorb β-TCP is not well understood. To elucidate this mechanism, we analyzed the structure of β-TCP discs on which mouse mature osteoclasts were cultured using scanning electron microscopy. We found that β-TCP was resorbed by mature osteoclasts on one side of each disc, as evidenced by the formation of multiple spine-like crystals at the exposed areas. Because osteoclasts secrete acid to resorb bone minerals, we mimicked this acidification by dipping β-TCP slices into HCl solution (pH 2.0). However, no spine-like crystals appeared even though the size of each β-TCP particle was reduced. On dentin slices, osteoclasts formed clear actin rings, which are cytoskeletal structures characteristic of bone-resorbing osteoclasts. No clear actin rings were observed in osteoclasts cultured on β-TCP slices, although small actin dots were observed. Analysis by transmission electron microscopy showed that osteoclasts attached to β-TCP particles. These results suggest that osteoclasts resorb β-TCP particles independently of clear actin ring formation.     

Parathyroid hormone-induced bone resorption does not occur in the absence of osteopontin

Osteopontin is an RGDS-containing protein that acts as a ligand for the alpha(v)beta(3) integrin, which is abundantly expressed in osteoclasts, cells responsible for bone resorption in osteopenic diseases such as osteoporosis and hyperparathyroidism. However, the role of osteopontin in the process of bone resorption has not yet been fully understood. Therefore, we investigated the direct function of osteopontin in bone resorption using an organ culture system. The amount of (45)Ca released from the osteopontin-deficient bones was not significantly different from the basal release from wild type bones. However, in contrast to the parathyroid hormone (PTH) enhancement of the (45)Ca release from wild type bones, PTH had no effect on (45)Ca release from organ cultures of osteopontin-deficient bones. Because PTH is located upstream of receptor activator of NF-kappaB ligand (RANKL), that directly promotes bone resorption, we also examined the effect of RANKL. Soluble RANKL with macrophage-colony stimulating factor enhanced (45)Ca release from the bones of wild type fetal mice but not from the bones of osteopontin-deficient mice. To obtain insight into the cellular mechanism underlying the phenomena observed in osteopontin-deficient bone, we investigated the number of tartrate-resistant acid phosphatase (TRAP)-positive cells in the bones subjected to PTH treatment in cultures. The number of TRAP-positive cells was increased significantly by PTH in wild type bone; however, no such PTH-induced increase in TRAP-positive cells was observed in osteopontin-deficient bones. These results indicate that the absence of osteopontin suppressed PTH-induced increase in bone resorption via preventing the increase in the number of osteoclasts in the local milieu of bone.