Osteoclast-bone Interface Research Articles

Linarin and its aglycone acacetin abrogate actin ring formation and focal contact to bone matrix of bone-resorbing osteoclasts through inhibition of αvβ3 integrin and core-linked CD44

BackgroundSince enhanced bone resorption due to osteoclast differentiation and activation cause skeletal diseases, there is a growing need in therapeutics for combating bone-resorbing osteoclasts. Botanical antioxidants are being increasingly investigated for their health-promoting effects on bone. Edible Cirsium setidens contains various polyphenols of linarin, pectolinarin, and apigenin with antioxidant and hepatoprotective effects. PurposeThis study aimed to determine whether linarin present in Cirsium setidens water extracts (CSE) and its aglycone acacetin inhibited osteoclastogenesis of RANKL-exposed RAW 264.7 murine macrophages for 5 days. MethodsThis study assessed the osteoprotective effects of CSE, linarin and acacetin on RANKL-induced differentiation and activation of osteoclasts by using MTT assay, TRAP staining, Western blot analysis, bone resorption assay actin ring staining, adhesion assay and immunocytochemical assay. This study explored the underlying mechanisms of their osteoprotection, and identified major components present in CSE by HPLC analysis. ResultsLinarin and pectolinarin were identified as major components of CSE. Nontoxic linarin and acacetin as well as CSE, but not pectolinarin attenuated the RANKL-induced macrophage differentiation into multinucleated osteoclasts, and curtailed osteoclastic bone resorption through reducing lacunar acidification and bone matrix degradation in the osteoclast-bone interface. Linarin and acacetin in CSE reduced the transmigration and focal contact of osteoclasts to bone matrix-mimicking RGD peptide. Such reduction was accomplished by inhibiting the induction of integrins, integrin-associated proteins of paxillin and gelsolin, cdc42 and CD44 involved in the formation of actin rings. The inhibition of integrin-mediated actin ring formation by linarin and acacetin entailed the disruption of TRAF6-c-Src-PI3K signaling of bone-resorbing osteoclasts. The functional inhibition of c-Src was involved in the loss of F-actin-enriched podosome core protein cortactin-mediated actin assembly due to linarin and acacetin. ConclusionThese observations demonstrate that CSE, linarin and acacetin were effective in retarding osteoclast function of focal adhesion to bone matrix and active bone resorption via inhibition of diffuse cloud-associated αvβ3 integrin and core-linked CD44.

Chondroitin Sulfate Promotes Activation of Cathepsin K

Cathepsin K (CatK), a major lysosomal collagenase produced by osteoclasts, plays an important role in bone resorption. Evidence exists that the collagenase activity of CatK is promoted by chondroitin sulfate (CS), a sulfated glycosaminoglycan. This study examines the role of CS in facilitating CatK activation. We have demonstrated that chondroitin 4-sulfate (C4-S) promotes autoprocessing of the pro-domain of CatK at pH ≤ 5, leading to a fully matured enzyme with collagenase and peptidase activities. We present evidence to demonstrate this autoactivation process is a trans-activation event that is efficiently inhibited by both the covalent cysteine protease inhibitor E-64 and the reversible selective CatK inhibitor L-006,235. During bone resorption, CatK and C4-S are co-localized at the ruffled border between osteoclast bone interface, supporting the proposal that CatK activation is accomplished through the combined action of the acidic environment together with the presence of a high concentration of C4-S. Formation of a multimeric complex between C4-S and pro-CatK has been speculated to accelerate CatK autoactivation and promote efficient collagen degradation. Together, these results demonstrate that CS plays an important role in contributing to the enhanced efficiency of CatK collagenase activity in vivo.

An SNX10 mutation causes malignant osteopetrosis of infancy

BackgroundOsteopetrosis is a life-threatening, rare disorder typically resulting from osteoclast dysfunction and infrequently from failure to commitment to osteoclast lineage. Patients commonly present in infancy with macrocephaly, feeding difficulties, evolving...

Vacuolar-type H+-ATPase-mediated acidosis promotes in vitro osteoclastogenesis via modulation of cell migration

Localized acidification of the osteoclast-bone interface is driven by a vacuolar-type H+-ATPase (V-ATPase) in the plasma membrane in a process thought to be associated with bone resorption. The present study investigated the mechanism underlying the roles of V-ATPase-induced acidosis in osteoclastogenesis. Active proton pumping due to increased V-ATPase activity during RANKL-induced osteoclastogenesis induced intracellular and extracellular acidification of osteoclast precursors. Subsequent analysis revealed blockage of extracellular acidification and induction of intracellular acidification by bafilomycin A1, a specific inhibitor of V-ATPase, indicating that extracellular acidification is mostly induced by V-ATPase-mediated proton pumping into extracellular space. Low-pH media controlled by HEPES-buffered conditions to mimic metabolic acidosis led to synergistic activation of RANKL-stimulated signals, including mitogen-activated protein kinases and transcription factor NF-kappaB, resulting in enhanced osteoclastogenesis. Low-pH media also upregulated the expression of osteopontin secreted into extracellular space, which is required for cell migration by binding to cell surface integrin alphavbeta3. Osteoclast precursor migration was significantly inhibited by treatment of antibodies to integrin alphavbeta3, resulting in the retardation of osteoclastogenesis. Taken together, these findings indicate that V-ATPase-driven acidosis modulates osteoclastogenesis.

The effect of fish oil on bone resorption following pulp exposure in rats.

The aim of this study was to determine whether treatment with fish oil may alter the periapical bone resorption following pulp exposure in rats. Untreated and pulp-exposed animals served as the negative and positive control, respectively. Other pulp-exposed animals were orally treated with fish oil at different concentrations and frequencies. Periapical tissue sections were stained with tartrate-resistant acid phosphatase (TRAP), and then the numbers of both periapical osteodasts and preosteoclasts were determined. The levels of bone resorption were assessed using the osteoclast-bone interface (OBI) index. The results showed that no differences between the numbers of both osteoclasts and preosteoclasts in the fish oil-treated animals and the negative control at day 14 could be found. Similarly, the periapical bone resorption in the animals treated with fish oil for 14 days significantly reduced to the levels of that in the negative control. The results of the present study, therefore, suggest that oral treatment with fish oil may inhibit bone resorption following pulp exposure in rats and hence, may have a therapeutic modality for inflamed periradicular tissue.

Osteoclastic acidification pathways during bone resorption

Osteoclasts resorb bone by attaching to the surface and then secreting protons into an extracellular compartment formed between osteoclast and bone surface. This secretion is necessary for bone mineral solubilization and the digestion of organic bone matrix by acid proteases. This study summarizes the characterization and role of each type of ion transport and defines the main biochemical mechanisms involved in the dissolution of bone mineral during bone resorption. The primary mechanism responsible for acidification of the osteoclast-bone interface is vacuolar H +-adenosine triphosphatase (ATPase) coupled with Cl − conductance localized to the ruffled membrane. Carbonic anhydrase II (CAII) provides the proton source for extracellular acidification by H +-ATPase and the HCO 3 − source for the HCO 3 −/Cl − exchanger. Whereas some transporters are responsible for the bone resorption process, others are essential for pH regulation in the osteoclast. The HCO 3 −/Cl − exchanger, in association with CAII, is the major transporter for maintenance of normal intracellular pH. An Na +/H + antiporter may also contribute to the recovery of intracellular pH during early osteoclast activation. Once this mechanism has been rendered inoperative, another conductive pathway translocates the protons and modulates cytoplasmic pH. Inward-rectifying K + channels may also be involved by compensating for the external acidification due to H + transport. These different effects of transport processes, either on bone resorption or pH homeostasis, increase the number of possible sites for pharmacological intervention in the treatment of metabolic bone diseases.

Scanning electrochemical microscopy at the surface of bone-resorbing osteoclasts: evidence for steady-state disposal and intracellular functional compartmentalization of calcium.

Osteoclast resorptive activity occurs despite the presence of extremely high levels of ionized calcium ([Ca2+]) within the osteoclast hemivacuole, which is generated as a by-product of its resorptive activity. Previous in vitro observations have shown that increases in extracellular [Ca2+] ([Ca2+]e) in the surrounding medium can inhibit the osteoclast resorptive activity. Therefore, it has been suggested that the osteoclast acts as a "sensor" for [Ca2+]e, and that high [Ca2+]e leads to an increase in intracellular [Ca2+] ([Ca2+]i), thereby inhibiting osteoclasts in a negative feedback manner. In this report we have carried out an experimental and theoretical analysis of calcium disposal during osteoclast activity to evaluate how in vitro models relate to in vivo osteoclast activity, where it is possible that high [Ca2+]e may be present in the hemivacuole but not over the nonresorbing surface of the cell. Scanning electrochemical microscopy (SECM) studies of [Ca2+] and superoxide anion (O2.-) generation by bone-resorbing osteoclasts on the surface of a bovine cortical bone slice were compared with microspectofluorometric measurements of the levels of [Ca2+]i in single osteoclasts and the effect of [Ca2+]i on various aspects of osteoclast function. The generation of O2.- by the osteoclasts has been shown to be positively correlated with osteoclast resorptive function and can therefore serve as an index of acute changes in osteoclast activity. The SECM of bone-resorbing osteoclasts at the surface of a bone slice revealed a continuous steady-state release of Ca2+. Even after prolonged incubation lasting 3 h the near-surface [Ca2+]e in the solution above the cell remained <2 mM. The SECM real-time measurement data were consistent with the osteoclast acting as a conduit for continuous Ca2+ disposal from the osteoclast-bone interface. We conclude that the osteoclast distinguishes [Ca2+]e in the hemivacuole and in the extracellular fluid above the cell which we denote [Ca2+]e. We found that an increase in [Ca2+]i may be associated with activation; inhibition; or be without effect on O2.- generation, bone-matrix, or bone resorption. Similarly, osteoclast adhesion and bone-resorbing activity was affected by [Ca2+]e' but showed no correlation with [Ca2+]i. The data suggest the existence of functional compartmentalization of [Ca2+]i within the osteoclast, where elevated calcium may have an inhibitory, excitatory, or no effect on the overall osteoclast activity while exerting a selective effect on different functional modalities. These observations lead to the conclusion that far from being inhibited by Ca2+ generated, the osteoclast by virtue of the observed functional compartmentalization is highly adapted at carrying out its activity even when the level of [Ca2+] in resorptive lacunae is elevated.

Immunohistochemical localization of membrane type 1-matrix metalloproteinase (MT1-MMP) in osteoclasts in vivo

Membrane type 1-matrix metalloproteinase (MT1-MMP) is capable of mediating proteolysis of extracellular matrix. The enzyme has been demonstrated in osteoclasts, in vitro. However, the precise localization in vivo, and therefore the function of the enzyme in osteoclasts, is still unclear. In this study, we immunohistochemically examined the localization of MT1-MMP in rat osteoclasts to clarify the role of MT1-MMP in osteoclastic bone resorption and bone turnover.The localization of MT1-MMP was visualized by the pre-embedding method using anti-MT1-MMP antibody and horseradish peroxidase (HRP) or gold-conjugated antibody. Immunoreactivity of anti-MT1-MMP was found in osteoclasts at the osteoclast–bone interface, but it was not uniform. Ultrastructurally, the immunoreactivity visualized by HRP was found in sealing zone. The plasma membrane at this site showed an irregular border and some invaginations. Immunoreactivity was also found on the surface of certain small vesicles in the cytoplasm. Enhanced silver granules were mainly associated with the sealing membrane.In this study, we demonstrated, for the first time, the localization of MT1-MMP in the sealing zone of osteoclastin vivo . Its distribution suggests that the enzyme modifies the bone surface to facilitate the migration and attachment of osteoclasts as well as scavenging the resorption lacunae.

Impact of the timing of indomethacin treatment in a model of synchronized bone remodelling in rats.

Prostaglandins (PGs) promote both bone resorption and formation in vitro and in vivo. In a synchronised model of bone remodelling, indomethacin, an inhibitor of PG synthesis, given from the start of the sequence, transiently impaired bone resorption. In this study we further explored the involvement of PGs in this model by treating rats with indomethacin (7.5 mg x kg(-1) x day(-1)) for 6 days from the peak of resorption (day 4 after activation in this model) or during reversal (day 6 after activation). In rats treated from day 4, the resorption surface (Oc.S/BS) and the number of osteoclasts (N.Oc/BPm) were higher on day 10 (+69 %, P < 0.01, and +60 %, P < 0.02 compared with controls, respectively); no effect on cell resorptive activity was observed. The bone formation surface (OS/BS) was reduced (-50 %, P < 0.01). The inactive surface (In/BS) was not modified. In rats treated from day 6, the Oc.S/BS was also higher than in controls (P < 0.02), as was the N.Oc/BPm (P < 0.05). Osteoclast activity appeared to be increased, as the osteoclast-bone interface was larger (P < 0.02), but the mean lacuna area was reduced (-23 %, P < 0.05). Bone formation was also strongly affected: the OS/BS was decreased (-66 %, P < 0.01), as was the osteoid seam thickness (-24 %, P < 0.05). The In/BS was increased 1.5-fold (P < 0.05). These data indicate that PGs intervene at various stages of this remodelling sequence, as both resorption and formation were affected by indomethacin. Although resorption resumed in the two treatment groups despite treatment continuation, the timing of treatment was clearly important. Only inhibition of PG synthesis at the peak of resorption delayed all phases of the remodelling sequence. In contrast, inhibition during the reversal phase prevented activation of a significant part of the bone surface usually involved at this stage of remodelling; this treatment schedule reduced the resorptive capacity of the system, and depressed osteoblast activity.

Chronic Extracellular Acidosis Induces Plasmalemmal Vacuolar Type H+ ATPase Activity in Osteoclasts

Proton extrusion into an extracellular resorption compartment is an essential component of bone degradation by osteoclasts. Chronic metabolic acidosis is known to induce negative calcium balance and bone loss by stimulating osteoclastic bone resorption, but the underlying mechanism is not known. The present studies were undertaken to evaluate whether chronic acidosis affects proton extrusion mechanisms in osteoclasts cultured on glass coverslips. Acidosis, mimicked experimentally by maintaining the cells at extracellular pH 6.5, rapidly lowered intracellular pH to 6.8. However, after 2 hours, a proportion of cells demonstrated the capacity to restore intracellular pH to near normal levels. To define the mechanism responsible for this recovery, the activity of individual H+ transport pathways was analyzed. We found that chronic acid treatment for up to 6 h did not significantly affect the cellular buffering power or Na+/H+ antiport activity. In contrast, chronic acidosis activated vacuolar H+ pumps in the osteoclasts. Although only approximately 5% of the control cells displayed proton pump activity, about 40% of cells kept at extracellular pH 6. 5 for 4-6 h were able to recover from the acute acid load by means of bafilomycin A1-sensitive proton extrusion. Conversely, the H+-selective conductance recently described in the plasma membrane of osteoclasts was clearly inhibited in the cells exposed to chronic acidosis. Following acid treatment, the activation threshold of the H+ conductance was shifted to more positive potentials, and the current density was significantly reduced. Considered together, these results suggest that induction of plasmalemmal vacuolar type ATPase activity by chronic acidosis, generated either systemically due to metabolic disease or locally at sites of inflammation, is likely to stimulate osteoclastic bone resorption and thus to promote bone loss.

Osteoclastic superoxide production and bone resorption: stimulation and inhibition by modulators of NADPH oxidase.

Production of superoxide radicals by osteoclasts is necessary for normal bone degradation. White blood cell superoxide, needed for bacterial killing, is produced by activated NADPH oxidase. Since osteoclasts and white blood cells share a common hematopoietic origin, we initiated experiments to test the hypothesis that superoxide radicals at the osteoclast-bone interface are produced by NADPH oxidase. Diphenyl iodonium (IDP), an inhibitor of NADPH oxidase, blocked superoxide generation and decreased osteoclastic bone resorption in cultures of calvarial explants from normal mice. Interferon (IFN) gamma, a stimulant of NADPH oxidase activity, increased superoxide production and bone resorption in cultures of calvarial explants from osteopetrotic (microphthalmic) mice. IDP blocked the stimulatory effects of IFN in this bone resorption model. These data suggest that osteoclastic superoxide is produced by NADPH oxidase.

Superoxide and bone resorption

The reaction of superoxide with nitroblue tetrazolium produces an electron-dense diformazan precipitate which can be used to localize areas of superoxide production. Transmission electron microscopy was used to demonstrate that difomazan granules formed by the reaction of nitroblue tetrazolium with excess superoxide are electron dense, whereas monoformazan granules generated by hydrogen peroxide were not. On the basis of these observations, superoxide formed along the osteoclast-bone interface was localized by demonstrating the electron-dense diformazan granules between the osteoclastic membrane and the bone surface. The formation of this reaction product was inhibited by a superoxide scavenger, the deferoxamine mesylate-manganese complex (the “green” complex), confirming the specificity of the reaction product. The scavenger also inhibited bone resorption. High concentrations of superoxide generated in vitro at a neutral pH degraded osteocalcin into numerous peptide fragments, demonstrating the ability of superoxide to break peptide bonds. These studies localize superoxide production to the ruffled border space and suggest that superoxide generated at the osteoclast-bone interface is involved in bone matrix degradation.

Partial inhibition of bone resorption by disodium cromoglycata in a synchronized model of bone remodeling

In a previous study we observed that mast cell degranulation might be associated to bone resorption. To verify this assumption, the efficiency of cromoglycate was assessed on a synchronized sequence of bone remodeling induced in male Wistar rats along the mandibular cortex. After 4 days (time of osteoclastic peak in this model), cromoglycate (15/mg/kg and 30 mg/kg/d per os) decreased the number of degranulating mast cells, especially in the population adjacent to the bone surface. Concomitantly the extent of resorption surface was decreased vs untreated animals. Active osteoclasts were lower whereas the total number of osteoclasts (both active and inactive) was not statistically modified. The mean osteoclast-bone interface was not modified. No dose effect was found. These data indicate that mast cell degranulation is involved in the events leading to osteoclast resorption, presumably in the mechanisms providing osteoclast access to bone surface.

The use of nitroblue-tetrazolium to measure the formation of superoxide in erythrocytes

The reaction of superoxide with nitroblue tetrazolium produces an electron-dense diformazan precipitate which can be used to localize areas of superoxide production. Transmission electron microscopy was used to demonstrate that difomazan granules formed by the reaction of nitroblue tetrazolium with excess superoxide are electron dense, whereas monoformazan granules generated by hydrogen peroxide were not. On the basis of these observations, superoxide formed along the osteoclast-bone interface was localized by demonstrating the electron-dense diformazan granules between the osteoclastic membrane and the bone surface. The formation of this reaction product was inhibited by a superoxide scavenger, the deferoxamine mesylate-manganese complex (the “green” complex), confirming the specificity of the reaction product. The scavenger also inhibited bone resorption. High concentrations of superoxide generated in vitro at a neutral pH degraded osteocalcin into numerous peptide fragments, demonstrating the ability of superoxide to break peptide bonds. These studies localize superoxide production to the ruffled border space and suggest that superoxide generated at the osteoclast-bone interface is involved in bone matrix degradation.

Oxygen derived free radicals in osteoclasts: The specificity and location of the nitroblue tetrazolium reaction

Oxygen derived free radicals are generated by osteoclasts. In a novel culture system, isolated rat osteoclasts were stained when nitroblue tetrazolium (NBT) was reduced by cellular oxidants to formazan, an insoluble precipitate. Superoxide dismutase (SOD) inhibited the accumulation of formazan by the isolated osteoclasts. Osteoclasts in mouse calvarial organ cultures also reduced NBT to formazan. The reaction products were localized to the area of the osteoclast-bone interface. At the light microscopic level, the formazan granules appeared to be concentrated within the cytoplasm. Formazan accumulation was significantly inhibited by calcitonin (hCT). The inhibition of NBT reduction by SOD indicates that the isolated osteoclasts were capable of producing superoxide. The localization of the formazan granules between the external osteoclastic membrane and the bone, and the inhibition of this reaction during hCT exposure suggests that oxygen derived free radicals may contribute to bone resorption.