Human macrophage colony-stimulating factor inhibits bone resorption by osteoclasts disaggregated from rat bone

Colony stimulating factors (CSFs) regulate the survival, proliferation and differentiation of haemopoietic progenitor cells, as well as the functional activity of mature cells. Because the osteoclast is derived from haemopoietic tissue, and because osteoblastic cells produce CSFs, we tested the effects of several CSFs on bone resorption by osteoclasts disaggregated from neonatal rat long bone. We found that recombinant macrophage (M)-CSF was a potent inhibitor of bone resorption, causing significant inhibition at concentrations similar to those required to support the growth of macrophage colonies in agar. Unlike other inhibitors of osteoclastic resorption, M-CSF did not alter cytoplasmic motility in time-lapse recordings, suggesting that M-CSF may inhibit osteoclasts through a different transduction mechanism. None of the remaining cytokines tested (granulocyte-macrophage CSF, interleukin 3, interleukin 6, or interferon γ) influenced bone resorption. M-CSF production may be a mechanism by which osteoblastic cells, which produce M-CSF, may regulate osteoclastic function. Alternatively, inhibition of osteoclastic resorption by a CSF that is responsible for amplification of the macrophage compartment may reflect a close lineage relationship between mononuclear phagocytes, in which M-CSF induces a diversion of lineage resources away from osteoclastic function.     

Stereophotogrammetric observations on bone resorption by isolated rabbit osteoclasts

Although the osteoclast has long been recognized as the major agent of bone resorption, it has previously only been possible to assess resorption by these cells in bone, where their response to environmental factors may be modified by, or even mediated by, the other cell types also present. We used a carborundum saw to prepare slices of human cortical bone which demonstrate a predictable surface appearance. Osteoclasts were disaggregated from neonatal rabbit long bone and settled onto these slices. After incubation for 24 hr we found that osteoclasts had formed distinctive excavations in the bone surface. This technique makes it possible, for the first time, to analyse the direct and indirect effects of hormones and cells on osteoclastic bone resorption.     

Dexamethasone inhibits bone resorption by indirectly inducing apoptosis of the bone-resorbing osteoclasts via the action of osteoblastic cells

Although glucocorticoids (GCs) are physiologically essentialfor bone metabolism, it is generally accepted that high dosesof GCs cause bone loss through a combination of decreased boneformation and increased bone resorption. However, the actionof GCs on mature osteoclasts remains contradictory. In thisstudy, we have examined the effect of GCs on osteoclasticbone-resorbing activity and osteoclast apoptosis, by using twodifferent cell types, rabbit unfractionated bone cells andhighly enriched mature osteoclasts (>95% of purity).Dexamethasone (Dex, 10^-10–10^-7 M) inhibited resorption pit formation on a dentine slice by the unfractionated bone cells in a dose- and time-dependent manner.However, Dex had no effect on the bone-resorbing activity of the isolated mature osteoclasts. When the isolated osteoclastswere co-cultured with rabbit osteoblastic cells, the osteoclastic bone resorption decreased in response to Dex,dependent on the number of osteoblastic cells. Like the effecton the bone resorption, Dex induced osteoclast apoptosis in cultures of the unfractionated bone cells, whereas it did not promote the apoptosis of the isolated osteoclasts. An inhibitorof caspases, Z-Asp-CH2-DCB attenuated both the inhibitory effecton osteoclastic bone resorption and the stimulatory effect onthe osteoclast apoptosis. In addition, the osteoblastic cellswere required for the osteoclast apoptosis induced by Dex. These findings indicate that the main target cells of GCs arenon-osteoclastic cells such as osteoblasts and that GCsindirectly inhibit bone resorption by inducing apoptosis ofthe mature osteoclasts through the action of non-osteoclasticcells. This study expands our knowledge about the multifunctional roles of GCs in bone metabolism.     

Tumor necrosis factor-alpha induces differentiation of and bone resorption by osteoclasts

Osteoclast progenitors differentiate into mature osteoclasts in the presence of receptor activator of NF-kappaB (RANK) ligand on stromal or osteoblastic cells and monocyte macrophage colony-stimulating factor (M-CSF). The soluble RANK ligand induces the same differentiation in vitro without stromal cells. Tumor necrosis factor-alpha (TNF-alpha), a potent cytokine involved in the regulation of osteoclast activity, promotes bone resorption via a primary effect on osteoblasts; however, it remains unclear whether TNF-alpha can also directly induce the differentiation of osteoclast progenitors into mature osteoclasts. This study revealed that TNF-alpha directly induced the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), which produced resorption pits on bone in vitro in the presence of M-CSF. The bone resorption activity of TNF-alpha-induced MNCs was lower than that of soluble RANK ligand-induced MNCs; however, interleukin-1beta stimulated this activity of TNF-alpha-induced MNCs without an increase in the number of MNCs. In this case, interleukin-1beta did not induce TRAP-positive MNC formation. The osteoclast progenitors expressed TNF receptors, p55 and p75; and the induction of TRAP-positive MNCs by TNF-alpha was inhibited completely by an anti-p55 antibody and partially by an anti-p75 antibody. Our findings presented here are the first to indicate that TNF-alpha is a crucial differentiation factor for osteoclasts. Our results suggest that TNF-alpha and M-CSF play an important role in local osteolysis in chronic inflammatory diseases.     

破骨细胞骨吸收功能的检测方法

破骨细胞是一种多核的,具有骨吸收功能的骨组织细胞,在骨吸收过程中起着至关重要的作用.破骨细胞骨吸收功能的异常会引发一系列的临床病症,如骨质疏松症、关节置换术后假体松动、骨硬化症和牙周病变等.破骨细胞骨吸收功能的进一步研究对于各类骨疾病的防治具有重要的意义.然而破骨细胞骨吸收功能的检测方法一直以来是制约破骨细胞研究的瓶颈之一.为此,围绕破骨细胞骨吸收功能的检测方法做一综述.     

Apoptotic bone cells may be engulfed by osteoclasts during alveolar bone resorption in young rats

The alveolar bone is a suitable in vivo physiological model for the study of apoptosis and interactions of bone cells because it undergoes continuous, rapid and intense resorption/remodelling, during a long period of time, to accommodate the growing tooth germs. The intensity of alveolar bone resorption greatly enhances the chances of observing images of the extremely rapid events of apoptosis of bone cells and also of images of interactions between osteoclasts and osteocytes/osteoblasts/bone lining cells. To find such images, we have therefore examined the alveolar bone of young rats using light microscopy, the TUNEL method for apoptosis, and electron microscopy. Fragments of alveolar bone from young rats were fixed in Bouin and formaldehyde for morphology and for the TUNEL method. Glutaraldehyde-formaldehyde fixed specimens were processed for transmission electron microscopy. Results showed TUNEL positive round/ovoid structures on the bone surface and inside osteocytic lacunae. These structures--also stained by hematoxylin--were therefore interpreted, respectively, as osteoblasts/lining cells and osteocytes undergoing apoptosis. Osteoclasts also exhibited TUNEL positive apoptotic bodies inside large vacuoles; the nuclei of osteoclasts, however, were always TUNEL negative. Ultrathin sections revealed typical apoptotic images--round/ ovoid bodies with dense crescent-like chromatin--on the bone surface, corresponding therefore to apoptotic osteoblasts/lining cells. Osteocytes also showed images compatible with apoptosis. Large osteoclast vacuoles often contained fragmented cellular material. Our results provide further support for the idea that osteoclasts internalize dying bone cells; we were however, unable to find images of osteoclasts in apoptosis.     

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.     

Effect of calcium ions on human calcitonin. Possible implications for bone resorption by osteoclasts

Calcium ions (Ca²⁺) are indispensable for life and are involved in important physiological actions, which makes maintaining a constant level of blood Ca²⁺ essential. Ca²⁺ is mainly stored in bones which serve as a reservoir and its homeostasis is modulated by various hormones. Human calcitonin (hCt) is a small peptide hormone that exerts its physiological effect on Ca²⁺ metabolism by means of osteoclast-mediated bone resorption inhibition. Most of these actions are mediated through peptide/receptor interaction that acts via a second messenger. However, in vitro studies have shown that hCt can interact with membrane lipids to form ion channels in membrane models. This ability is due to the peptide’s secondary structure and aggregation state, that can be modulated by different molecules. In our study, we evaluated the effect of Ca²⁺, at different concentrations, both on the hCt ion channel incorporated into a planar lipid membrane made up of phosphatidylcholine containing 15 % phosphatidylglycerol and on the secondary structure of hCt in an aqueous environment. Ca²⁺ is able to interact with the hCt peptide by acting on the channel incorporated into the membrane as well as on the peptide in solution, both by increasing hCt channel frequency and in solution promoting α-helix formation, that counteracts the fibrillating process. These experimental observations, suggesting that hCt senses Ca²⁺ concentration variations, strengthen the hypothesis that channel formation represents an extra source of Ca²⁺ entry into osteoclasts in addition to the well-known interaction of the monomer with the specific receptor.     

Tamoxifen inhibits phorbol ester stimulated osteoclastic bone resorption: an effect mediated by calmodulin.

Tamoxifen inhibits bone resorption by disrupting calmodulin-dependent processes. Since tamoxifen inhibits protein kinase C in other cells, we compared the effects of tamoxifen and the phorbol ester, phorbol myristate acetate, on osteoclast activity. Phorbol esters stimulate bone resorption and calmodulin levels four-fold (k0.5 = 0.1-0.3 microM). In contrast, tamoxifen inhibited osteoclast activity approximately 60% with an IC50 of 1.5 microM, had no apparent effect on protein kinase C activity in whole-cell lysates, and reduced protein kinase C alpha recovered by immunoprecipitation 75%. Phorbol esters stimulated resorption in a time-dependent manner that was closely correlated with a similar-fold increase in calmodulin. Protein kinase C alpha, beta, delta, epsilon, and zeta were all down-regulated in response to phorbol ester treatment. Tamoxifen and trifluoperazine inhibited PMA-dependent increases in bone resorption and calmodulin by 85 +/- 10%. Down-regulation of protein kinase C isoforms by phorbol esters suggests that the observed increases in bone resorption and calmodulin levels are most likely due to a mechanism independent of protein kinase C and dependent on calmodulin. In conclusion, the data suggest that protein kinase C negatively regulates calmodulin expression and support the hypothesis that the effects of both phorbol esters and tamoxifen on osteoclast activity is mediated by calmodulin.     

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

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

Ametantrone inhibits prostaglandin--mediated resorption in bone organ culture

Prostaglandins (PG) have been postulated to be involved in both tumor metastases to bone and in tumor-induced bone resorption. The anthracenedione antineoplastic agents ametantrone (HAQ) and mitoxantrone are potent antioxidants and inhibit hydroperoxide-dependent initiation and propagation reactions. Therefore, these compounds may inhibit PG production and could also inhibit tumor metastases and tumor-induced resorption. The ability of HAQ, a prototypic anthracenedione, to inhibit PG synthesis and PG-mediated bone resorption was investigated using neonatal mouse calvaria in organ culture. Epidermal growth factor (EGF) stimulates bone resorption in this tissue by inducing PG synthesis. Consequently, if HAQ inhibits EGF-stimulated PG synthesis, it should also inhibit EGF-stimulated bone resorption. HAQ, at 10 microM, completely abolished EGF-stimulated PG synthesis and calcium release. Moreover, HAQ (1.0-30 microM) inhibition of EGF-stimulated PGE2 synthesis correlated with the inhibition of EGF-stimulated Ca release in a concentration-dependent manner. In contrast to EGF, parathyroid hormone stimulates resorption by a PG-independent pathway. HAQ at 10 microM had no effect on parathyroid hormone stimulated Ca release. These results suggest that HAQ inhibition of bone resorption appears to be primarily mediated by inhibition of PG biosynthesis.     

Divalent cations mimic the inhibitory effect of extracellular ionised calcium on bone resorption by isolated rat osteoclasts: further evidence for a "calcium receptor".

Osteoclast activity is thought to be regulated by calcitonin, as well as by the level of ionised calcium generated locally as a result of bone resorption. The exposure of isolated osteoclasts to elevated ambient calcium levels has been shown to lower resorptive activity and to reduce rates of enzyme release. We have attempted to determine whether these effects are mediated by a divalent cation-sensitive "calcium receptor," as has been reported for the parathyroid chief cells. Thus, we compared the effect of alkaline earth metal cations on osteoclast function using a morphometric measure of bone resorption and a spectrophotometric method for measuring the activity of the released enzyme, acid phosphatase. The exposure of resorbing osteoclasts to between 5 and 20 mM extracellular ionised calcium ([Ca2+]e) inhibited bone resorption and enzyme release to an extent similar to that seen with 0.1 to 10 microM ionomycin. The effect of combining submaximal concentrations of [Ca2+]e (15 mM) and ionomycin (0.1 microM) resulted in additivity, suggesting that the influence of [Ca2+]e on bone resorption was mediated by elevated intracellular calcium levels ([Ca2+]i). The other cations studied (Mg2+, Ba2+) were effective and elicited similar effects, although some required higher concentrations. Thus, whilst Ca2+ and Mg2+ were effective at 10 to 15 mM levels, Ba2+ was effective only at high (20 mM) concentrations. These findings are consistent with an influence of [Ca2+]e on osteoclast activity through an action on a surface membrane "calcium receptor" that can also bind other divalent cations, rather than by passive changes of [Ca2+]i with [Ca2+]e elevation.     

Bone acidic glycoprotein 75 inhibits resorption activity of isolated rat and chicken osteoclasts.

Matrix protein effects on the differentiated activity of osteoclasts were examined in order to understand the functional significance of bone protein interactions with osteoclasts. Bone acidic glycoprotein 75 (BAG 75) from rat calvariae inhibited the resorption of bone by isolated rat osteoclasts with IC50 = 1 nM compared to IC50 = 10 nM for chicken osteoclasts. By contrast, other phosphoproteins similarly isolated from bone were less effective in inhibiting resorption with IC50 = 100 nM osteopontin and IC50 greater than 100 nM bone sialoprotein. Likewise, RGD-containing matrix proteins vitronectin, thrombospondin, and fibronectin all displayed IC50 greater than or equal to 100 nM. Mechanistically, 10 nM BAG 75 marginally slowed, but did not block, the association of bone particles with chicken osteoclasts compared with osteopontin or control media. Pretreatment of osteoclasts with 50 nM BAG 75 had no effect on subsequent bone resorption; however, pretreatment of bone with BAG 75 before incubation with osteoclasts reduced the extent of resorption by 55%. These data suggest that a BAG 75/bone surface complex, rather than BAG 75 alone, represents the inhibitory form. Consistent with this hypothesis, direct binding studies provided no evidence of specific, high-affinity receptors on osteoclasts for BAG 75, nor was an excess of BAG 75 (100 nM) able to compete with 0.3 nM sechistatin for osteoclastic avB3-like receptors. However, BAG 75 displayed cooperative binding to tissue fragments and bone particles at concentrations greater than 10 nM, suggesting that BAG 75 self-associates into higher-order species on bone surfaces. Electron microscopy confirmed the time-dependent polymerization of BAG 75 into interconnecting filaments. These data suggest a novel, inhibitory activity for surface-bound BAG 75 on bone resorption that does not appear to involve the osteoclastic avB3-like integrin.     

alpha-Lipoic acid inhibits inflammatory bone resorption by suppressing prostaglandin E2 synthesis

alpha-Lipoic acid (LA) has been intensely investigated as a therapeutic agent for several pathological conditions, including diabetic polyneuropathy. In the present study, we examined the effects of LA on osteoclastic bone loss associated with inflammation. LA significantly inhibited IL-1-induced osteoclast formation in cocultures of mouse osteoblasts and bone marrow cells, but LA had only a marginal effect on osteoclastogenesis from bone marrow macrophages induced by receptor activator of NF-kappaB ligand (RANKL). LA inhibited both the sustained up-regulation of RANKL expression and the production of PGE2 induced by IL-1 in osteoblasts. In addition, treatment with either prostaglandin E2 (PGE2) or RANKL rescued IL-1-induced osteoclast formation inhibited by LA or NS398, a specific cyclooxygenase-2 (COX-2) inhibitor, in cocultures. LA blocked IL-1-induced PGE2 production even in the presence of arachidonic acid, without affecting the expression of COX-2 and membrane-bound PGE2 synthase. Dihydrolipoic acid (the reduced form of LA), but not LA, attenuated recombinant COX-2 activity in vitro. LA also inhibited osteoclast formation and bone loss induced by IL-1 and LPS in mice. Our results suggest that the reduced form of LA inhibits COX-2 activity, PGE2 production, and sustained RANKL expression, thereby inhibiting osteoclast formation and bone loss in inflammatory conditions.     

Dissociation of bone resorption and bone formation in adult mice with a non-functional V-ATPase in osteoclasts leads to increased bone strength

Osteopetrosis caused by defective acid secretion by the osteoclast, is characterized by defective bone resorption, increased osteoclast numbers, while bone formation is normal or increased. In contrast the bones are of poor quality, despite this uncoupling of formation from resorption.To shed light on the effect of uncoupling in adult mice with respect to bone strength, we transplanted irradiated three-month old normal mice with hematopoietic stem cells from control or oc/oc mice, which have defective acid secretion, and followed them for 12 to 28 weeks.Engraftment levels were assessed by flow cytometry of peripheral blood. Serum samples were collected every six weeks for measurement of bone turnover markers. At termination bones were collected for μCT and mechanical testing. An engraftment level of 98% was obtained. From week 6 until termination bone resorption was significantly reduced, while the osteoclast number was increased when comparing oc/oc to controls. Bone formation was elevated at week 6, normalized at week 12, and reduced onwards. μCT and mechanical analyses of femurs and vertebrae showed increased bone volume and bone strength of cortical and trabecular bone.In conclusion, these data show that attenuation of acid secretion in adult mice leads to uncoupling and improves bone strength.     

Functional heterogeneity of osteoclasts: matrix metalloproteinases participate in osteoclastic resorption of calvarial bone but not in resorption of long bone.

Data in the literature suggest that site-specific differences exist in the skeleton with respect to digestion of bone by osteoclasts. Therefore, we investigated whether bone resorption by calvarial osteoclasts (intramembranous bone) differs from resorption by long bone osteoclasts (endochondral bone). The involvement of two major classes of proteolytic enzymes, the cysteine proteinases (CPs) and matrix metalloproteinases (MMPs), was studied by analyzing the effects of selective low molecular weight inhibitors of these enzymes on bone resorption. Mouse tissue explants (calvariae and long bones) as well as rabbit osteoclasts, which had been isolated from both skeletal sites and subsequently seeded on bone slices, were cultured in the presence of inhibitors and resorption was analyzed. The activity of the CP cathepsins B and K and of MMPs was determined biochemically (CPs and MMPs) and enzyme histochemically (CPs) in explants and isolated osteoclasts. We show that osteoclastic resorption of calvarial bone depends on activity of both CPs and MMPs, whereas long bone resorption depends on CPs, but not on the activity of MMPs. Furthermore, significantly higher levels of cathepsin B and cathepsin K activities were expressed by long bone osteoclasts than by calvarial osteoclasts. Resorption of slices of bovine skull or cortical bone by osteoclasts isolated from long bones was not affected by MMP inhibitors, whereas resorption by calvarial osteoclasts was inhibited. Inhibition of CP activity affected the resorption by the two populations of osteoclasts in a similar way. We conclude that this is the first report to show that significant differences exist between osteoclasts of calvariae and long bones with respect to their bone resorbing activities. Resorption by calvarial osteoclasts depends on the activity of CPs and MMPs, whereas resorption by long bone osteoclasts depends primarily on the activity of CPs. We hypothesize that functionally different subpopulations of osteoclasts, such as those described here, originate from different sets of progenitors.     

V-ATPases in osteoclasts: structure, function and potential inhibitors of bone resorption

The vacuolar-type H(+)-ATPase (V-ATPase) proton pump is a macromolecular complex composed of at least 14 subunits organized into two functional domains, V(1) and V(0). The complex is located on the ruffled border plasma membrane of bone-resorbing osteoclasts, mediating extracellular acidification for bone demineralization during bone resorption. Genetic studies from mice to man implicate a critical role for V-ATPase subunits in osteoclast-related diseases including osteopetrosis and osteoporosis. Thus, the V-ATPase complex is a potential molecular target for the development of novel anti-resorptive agents useful for the treatment of osteolytic diseases. Here, we review the current structure and function of V-ATPase subunits, emphasizing their exquisite roles in osteoclastic function. In addition, we compare several distinct classes of V-ATPase inhibitors with specific inhibitory effects on osteoclasts. Understanding the structure-function relationship of the osteoclast V-ATPase may lead to the development of osteoclast-specific V-ATPase inhibitors that may serve as alternative therapies for the treatment of osteolytic diseases.     

The in vitro effect of pH on osteoclasts and bone resorption in the cat: implications for the pathogenesis of FORL

Dental disease due to osteoclast over-activity reaches epidemic proportions in older domestic cats and has also been reported in wild cats. Feline osteoclastic resorptive lesions (FORL) involve extensive resorption of the tooth leaving it liable to root fracture and subsequent tooth loss. The aetio-pathogenesis of FORL is not known. Recent work has shown that systemic acidosis causes increased osteoclast activation and that loci of infection or inflammation in cat mouth are likely to be acidotic. To investigate this, we generated osteoclasts from cat blood and found that they formed in large numbers (approximately 400) in cultures on bovine cortical bone slices. Acidosis caused an increase in the size of cells-in cultures maintained up to 14 days at basal pH 7.25, mean osteoclast area was 0.01 +/- 0.003 mm(2), whereas an 8.6-fold increase was observed in cells cultured between 11 and 14 days at pH 7.15 (0.086 +/- 0.004 mm(2)). Acidosis caused a modest increase in the number of osteoclasts. Exposure to pH 6.92 exhibited a 5-fold increase in the area of bone slices covered by resorption lacunae ( approximately 70% bone slice resorbed). In line with this finding, significant increases were observed in the expression of cathepsin K and proton pump enzymes (both approximately 3-fold) that are key enzymes reflective of resorptive activity in osteoclasts. These results demonstrate that acidosis is a major regulator of osteoclast formation and functional activation in the cat, and suggest that local pH changes may play a significant role in the pathogenesis of FORL.     

Localization of rat cathepsin K in osteoclasts and resorption pits: inhibition of bone resorption and cathepsin K-activity by peptidyl vinyl sulfones.

We have localized cathepsin K in rat osteoclasts and within exposed resorption pits by immuno-fluorescence microscopy. Intracellular staining using an antibody raised against recombinant mouse cathepsin K was vesicular and uniformly distributed throughout the cell. Confocal microscopy analysis did not reveal an accumulation of cathepsin K containing vesicles opposing the ruffled border and the resorption lacuna. Exposed resorption pits exhibited a uniform distribution of cathepsin K, and no differences were observed between the edges and the centers of the pits. The immunostaining of resorption pits with anti-cathepsin K antibodies demonstrates that the protease is secreted into the sub-osteoclastic compartment. Cathepsin K-specific inhibition using peptidyl vinyl sulfones as selective cysteine protease inactivators reduced bone resorption by 80% in a dose-dependent manner at sub-micromolar concentrations. No reduction of bone resorption was observed at those low concentrations using a potent cathepsin L, S, B-specific inhibitor. That the inhibition of bone resorption can be attributed to cathepsin K-like protease inhibition was corroborated by the selective inhibition of the osteoclastic Z-Gly-Pro-Arg-MbetaNA hydrolyzing activity by the cathepsin K, L, S, B-inhibitor, but not by the cathepsin L, B, and S inhibitor. Z-Gly-Pro-Arg-MbetaNA is efficiently hydrolyzed by cathepsin K but only poorly by cathepsins L, S, and B. On the contrary, the intracellular hydrolysis of the cathepsin B-specific substrate, Z-Arg-Arg-MbetaNA, was prevented by both types of inhibitors. The identification of cathepsin K in resorption pits and the inhibition of bone resorption and intracellular cathepsin K activity by selective vinyl sulfone inhibitors indicate the critical role of the protease in osteoclastic bone resorption.