Osteoclastic bone degradation and the role of different cysteine proteinases and matrix metalloproteinases: differences between calvaria and long bone.

Osteoclastic bone degradation involves the activity of cathepsin K. We found that in addition to this enzyme other, yet unknown, cysteine proteinases participate in digestion. The results support the notion that osteoclasts from different bone sites use different enzymes to degrade the collagenous bone matrix. INTRODUCTION: The osteoclast resorbs bone by lowering the pH in the resorption lacuna, which is followed by secretion of proteolytic enzymes. One of the enzymes taken to be essential in resorption is the cysteine proteinase, cathepsin K. Some immunolabeling and enzyme inhibitor data, however, suggest that other cysteine proteinases and/or proteolytic enzymes belonging to the group of matrix metalloproteinases (MMPs) may participate in the degradation. In this study, we investigated whether, in addition to cathepsin K, other enzymes participate in osteoclastic bone degradation. MATERIALS AND METHODS: In bones obtained from mice deficient for cathepsin K, B, or L or a combination of K and L, the bone-resorbing activity of osteoclasts was analyzed at the electron microscopic level. In addition, bone explants were cultured in the presence of different selective cysteine proteinase inhibitors and an MMP inhibitor, and the effect on resorption was assessed. Because previous studies showed differences in resorption by calvarial osteoclasts compared with those present in long bones, in all experiments, the two types of bone were compared. Finally, bone extracts were analyzed for the level of activity of cysteine proteinases and the effect of inhibitors hereupon. RESULTS: The analyses of the cathepsin-deficient bone explants showed that, in addition to cathepsin K, calvarial osteoclasts use other cysteine proteinases to degrade bone matrix. It was also shown that, in the absence of cathepsin K, long bone osteoclasts use MMPs for resorption. Cathepsin L proved to be involved in the MMP-mediated resorption of bone by calvarial osteoclasts; in the absence of this cathepsin, calvarial osteoclasts do not use MMPs for resorption. Selective inhibitors of cathepsin K and other cysteine proteinases showed a stronger effect on calvarial resorption than on long bone resorption. CONCLUSIONS: Our findings suggest that (1) cathepsin K-deficient long bone osteoclasts compensate the lack of this enzyme by using MMPs in the resorption of bone matrix; (2) cathepsin L is involved in MMP-mediated resorption by calvarial osteoclasts; (3) in addition to cathepsin K, other, yet unknown, cysteine proteinases are likely to participate in skull bone degradation; and finally, (4) the data provide strong additional support for the existence of functionally different bone-site specific osteoclasts.     

Expression of Cathepsins B, H, K, L, and S and Matrix Metalloproteinases 9 and 13 During Chondrocyte Hypertrophy and Endochondral Ossification in Mouse Fracture Callus

Fracture repair provides an interesting model for chondrogenesis and osteogenesis as it recapitulates in an adult organism the same steps encountered during embryonic skeletal development and growth. The fracture callus is not only a site of rapid production of cartilage and bone, but also a site of extensive degradation of their extracellular matrices. The present study was initiated to increase our understanding of the roles of different proteolytic enzymes, cysteine cathepsins B, H, K, L, and S, and matrix metalloproteinases (MMPs) 9 and 13, during fracture repair, as this aspect of bone repair has previously received little attention. Northern analysis revealed marked upregulation of cathepsin K, MMP-9, and MMP-13 mRNAs during the first and second weeks of healing. The expression profiles of these mRNAs were similar with that of osteoclastic marker enzyme tartrate-resistant alkaline phosphatate (TRAP). The changes in the mRNA levels of cathepsins B, H, L, and S were smaller when compared with those of the other enzymes studied. Immunohistochemistry and in situ hybridization confirmed the predominant localization of cathepsin K and MMP-9 and their mRNA in osteoclasts and chondroclasts at the osteochondral junction. MMP-13 was present in osteoblasts and individual hypertrophic chondrocytes near the cartilage-bone interphase. In cartilaginous callus, the expression of cathepsins B, H, L, and S was mainly related to chondrocyte hypertrophy. During bone remodeling both osteoblasts and osteoclasts contained these cathepsins. The present data demonstrate that degradation and remodeling of extracellular matrices during fracture healing involves activation of MMP-13 production in hypertrophic chondrocytes and osteoblasts, and cathepsin K and MMP-9 production in osteoclasts and chondroclasts. Received: 2 February 2000 / Accepted: 25 May 2000 / Online publication: 2 November 2000     

An ultrastructural evaluation of the effects of cysteine-proteinase inhibitors on osteoclastic resorptive functions

This study was designed to evaluate the effects of specific and potent cathepsin inhibitors on osteoclastic resorptive functions in vitro by means of a novel ultrastructural assay system. Mouse bone marrow cell-derived osteoclasts were suspended on dentine slices and cultured for 48 hours in the presence of either E-64 (a generalized cysteine proteinase inhibitor) or Z-Phe-Phe-CHN₂ (a selective cathepsin L inhibitor). After the removal of cultured osteoclasts, co-cultured dentine slices were examined using electron microscopy: backscattered (BSEM), scanning (SEM), and atomic force (AFM). In morphometric analyses of BSEM images, there were no significant differences in the areas of demineralized dentine surfaces between control and inhibitor-treated groups, suggesting that cathepsin inhibitors had no effect on dentine demineralization by cultured osteoclasts. However, in SEM and AFM observations, both inhibitors remarkably reduced to the same extent, the formation of deep resorption lacunae on dentine slices that had resulted from degradation of matrix collagen. In addition, Z-Phe-Phe-CHN₂ treatment produced deeper, ring-like grooves with little collagen exposure in shallow resorption lacunae. These results strongly suggest that (1) cathepsins released by osteoclasts are involved in the formation of deep resorption lacunae, and (2) cathepsin L plays a key role in bone resorption.     

Different Cysteine Proteinases Involved in Bone Resorption and Osteoclast Formation

Cysteine proteinases, especially cathepsin K, play an important role in osteoclastic degradation of bone matrix proteins and the process can, consequently, be significantly inhibited by cysteine proteinase inhibitors. We have recently reported that cystatin C and other cysteine proteinase inhibitors also reduce osteoclast formation. However, it is not known which cysteine proteinase(s) are involved in osteoclast differentiation. In the present study, we compared the relative potencies of cystatins C and D as inhibitors of bone resorption in cultured mouse calvariae, osteoclastogenesis in mouse bone marrow cultures, and cathepsin K activity. Inhibition of cathepsin K activity was assessed by determining equilibrium constants for inhibitor complexes in fluorogenic substrate assays. The data demonstrate that whereas human cystatins C and D are equipotent as inhibitors of bone resorption, cystatin D is 10-fold less potent as an inhibitor of osteoclastogenesis and 200-fold less potent as an inhibitor of cathepsin K activity. A recombinant human cystatin C variant with Gly substitutions for residues Arg⁸, Leu⁹, Val¹⁰, and Trp¹⁰⁶ did not inhibit bone resorption, had 1,000-fold decreased inhibitory effect on cathepsin K activity compared to wildtype cystatin C, but was equipotent with wildtype cystatin C as an inhibitor of osteoclastogenesis. It is concluded that (i) different cysteine proteinases are likely to be involved in bone resorption and osteoclast formation, (ii) cathepsin K may not be an exclusive target enzyme in any of the two systems, and (iii) the enzyme(s) involved in osteoclastogenesis might not be a typical papain-like cysteine proteinase.     

Calvarial Osteoclasts Express a Higher Level of Tartrate-Resistant Acid Phosphatase than Long Bone Osteoclasts and Activation Does not Depend on Cathepsin K or L Activity

Bone resorption by osteoclasts depends on the activity of various proteolytic enzymes, in particular those belonging to the group of cysteine proteinases. Next to these enzymes, tartrate-resistant acid phosphatase (TRAP) is considered to participate in this process. TRAP is synthesized as an inactive proenzyme, and in vitro studies have shown its activation by cysteine proteinases. In the present study, the possible involvement of the latter enzyme class in the in vivo modulation of TRAP was investigated using mice deficient for cathepsin K and/or L and in bones that express a high (long bone) or low (calvaria) level of cysteine proteinase activity. The results demonstrated, in mice lacking cathepsin K but not in those deficient for cathepsin L, significantly higher levels of TRAP activity in long bone. This higher activity was due to a higher number of osteoclasts. Next, we found considerable differences in TRAP activity between calvarial and long bones. Calvarial bones contained a 25-fold higher level of activity than long bones. This difference was seen in all mice, irrespective of genotype. Osteoclasts isolated from the two types of bone revealed that calvarial osteoclasts expressed higher enzyme activity as well as a higher level of mRNA for the enzyme. Analysis of TRAP-deficient mice revealed higher levels of nondigested bone matrix components in and around calvarial osteoclasts than in long bone osteoclasts. Finally, inhibition of cysteine proteinase activity by specific inhibitors resulted in increased TRAP activity. Our data suggest that neither cathepsin K nor L is essential in activating TRAP. The findings also point to functional differences between osteoclasts from different bone sites in terms of participation of TRAP in degradation of bone matrix. We propose that the higher level of TRAP activity in calvarial osteoclasts compared to that in long bone cells may partially compensate for the lower cysteine proteinase activity found in calvarial osteoclasts and TRAP may contribute to the degradation of noncollagenous proteins during the digestion of this type of bone. An erratum to this article is available at .     

Comparison in localization between cystatin C and cathepsin K in osteoclasts and other cells in mouse tibia epiphysis by immunolight and immunoelectron microscopy

We compared the distribution of a cysteine proteinase inhibitor, cystatin C, with that of cathepsin K in osteoclasts of the mouse tibia by immunolight and immunoelectron microscopy. Light microscopically, strong immunoreactivity for cystatin C was found extracellularly along the resorption lacuna and intracellularly in the organelles of osteoclasts. In serial sections, various patterns of cystatin C and cathepsin K localization were seen, specifically: (1) some resorption lacuna were positive for both cystatin C and cathepsin K; (2) others were positive for either cystatin C or cathepsin K, but not both; and (3) some lacuna were negative for both. In osteoclasts, the localization of cystatin C was similar to that of cathepsin K. Furthermore, cystatin C immunoreactivity was detected in preosteoclasts and osteoblasts, whereas cathepsin K was seen only in preosteoclasts. Electron microscopically, cystatin C immunoreactive products were found in the rough endoplasmic reticulum (ER), Golgi apparatus, vesicles, granules, and vacuoles of osteoclasts. These cystatin C-positive vesicles had fused or were in the process of fusion with the ampullar vacuoles (extracellular spaces) containing cystatin C-positive, fragmented, fibril-like structures. The extracellular cystatin C was deposited on and between the cytoplasmic processes of ruffled borders, and on and between type I collagen fibrils. In the basolateral region of osteoclasts, cystatin C-positive vesicles and granules also fused with vacuoles that contained cystatin C-positive or negative fibril-like structures. These results indicate that osteoclasts not only synthesize and secrete cathepsin K from the ruffled border into the bone resorption lacunae, but also a cysteine proteinase inhibitor, cystatin C. Therefore, it is suggested that cystatin C regulates the degradation of bone matrix by cathepsin K, both extracellularly and intracellularly.     

A highly potent inhibitor of cathepsin K (relacatib) reduces biomarkers of bone resorption both in vitro and in an acute model of elevated bone turnover in vivo in monkeys

Cathepsin K is an osteoclast-derived cysteine protease that has been implicated as playing a major role in bone resorption. A substantial body of evidence indicates that cathepsin K is critical in osteoclast-mediated bone resorption and suggests that its pharmacological inhibition should result in inhibition of bone resorption in vivo. Here we report the pharmacological characterization of SB-462795 (relacatib) as a potent and orally bioavailable small molecule inhibitor of cathepsin K that inhibits bone resorption both in vitro in human tissue and in vivo in cynomolgus monkeys. SB-462795 is a potent inhibitor of human cathepsins K, L, and V (K(i, app)=41, 68, and 53 pM, respectively) that exhibits 39-300-fold selectivity over other cathepsins. SB-462795 inhibited endogenous cathepsin K in situ in human osteoclasts and human osteoclast-mediated bone resorption with IC50 values of approximately 45 nM and approximately 70 nM, respectively. The anti-resorptive potential of SB-462795 was evaluated in normal as well as medically ovariectomized (Ovx) female cynomolgus monkeys. Serum levels of the C- and N-terminal telopeptides of Type I collagen (CTx and NTx, respectively) and urinary levels of NTx were monitored as biomarkers of bone resorption. Administration of SB-462795 to medically ovariectomized or normal monkeys resulted in an acute reduction in both serum and urinary markers of bone resorption within 1.5 h after dosing, and this effect lasted up to 48 h depending on the dose administered. Our data indicate that SB-462795 potently inhibits human cathepsin K in osteoclasts, resulting in a rapid inhibition of bone resorption both in vitro and in vivo in the monkey. These studies also demonstrate the therapeutic potential of relacatib in the treatment of postmenopausal osteoporosis and serves to model the planned clinical trials in human subjects.     

Proteolytic processing and polarized secretion of tartrate-resistant acid phosphatase is altered in a subpopulation of metaphyseal osteoclasts in cathepsin K-deficient mice

Tartrate-resistant acid phosphatase (TRAP) is an enzyme highly expressed in osteoclasts and thought to participate in osteoclast-mediated bone turnover. Cathepsin K (Ctsk) is the major collagenolytic cysteine proteinase expressed in osteoclasts and has recently been shown to be able to proteolytically process and activate TRAP in vitro. In this study, 4-week-old Ctsk(-/-) mice were analysed for TRAP expression at the mRNA, protein and enzyme activity levels to delineate a role of cathepsin K in TRAP processing in osteoclasts in vivo. The absence of cathepsin K in osteoclasts was associated with increased expression of TRAP mRNA, monomeric TRAP protein and total TRAP activity. Proteolytic processing of TRAP was not abolished but prematurely arrested at an intermediate stage without changing enzyme activity, a finding confirmed with RANKL-differentiated osteoclast-like cell line RAW264.7 treated with the cysteine proteinase inhibitor E-64. Thus, the increase in total TRAP activity was mainly due to increased cellular content of monomeric TRAP. The increase in monomeric TRAP expression was more pronounced in osteoclasts of the distal compared to the proximal part of the metaphyseal trabecular bone, suggesting a site-dependent role for cathepsin K in TRAP processing. Moreover, intracellular localization of monomeric TRAP was altered in distal metaphyseal osteoclasts from Ctsk(-/-) mice. Additionally, TRAP was secreted into the ruffled border as the processed form in osteoclasts of Ctsk(-/-) mice, unlike in osteoclasts from wild-type mice which secreted TRAP to the resorption lacuna as the monomeric form. The results demonstrate that cathepsin K is not only involved in proteolytic processing but also affects the intracellular trafficking of TRAP, particularly in osteoclasts of the distal metaphysis. However, contribution by other yet unidentified protease(s) to TRAP processing must also be invoked since proteolytic cleavage of TRAP is not abolished in Ctsk(-/-) mice. Importantly, this study highlights functional differences between bone-resorbing clasts within the trabecular metaphyseal bone, suggesting potentially important differences in the regulation of differentiation and activation depending on the precise anatomical localization of the clast population.     

Human osteoclast cathepsin K is processed intracellularly prior to attachment and bone resorption.

Cathepsin K is a member of the papain superfamily of cysteine proteases and has been proposed to play a pivotal role in osteoclast-mediated bone resorption. We have developed a sensitive cytochemical assay to localize and quantify osteoclast cathepsin K activity in sections of osteoclastoma and human bone. In tissue sections, osteoclasts that are distant from bone express high levels of cathepsin K messenger RNA (mRNA) and protein. However, the majority of the cathepsin K in these cells is in an inactive zymogen form, as assessed using both the cytochemical assay and specific immunostaining. In contrast, osteoclasts that are closer to bone contain high levels of immunoreactive mature cathepsin K that codistributes with enzyme activity in a polarized fashion toward the bone surface. Polarization of active enzyme was clearly evident in osteoclasts in the vicinity of bone. The osteoclasts apposed to the bone surface were almost exclusively expressing the mature form of cathepsin K. These cells showed intense enzyme activity, which was polarized at the ruffled border. These results suggest that the in vivo activation of cathepsin K occurs intracellularly, before secretion into the resorption lacunae and the onset of bone resorption. The processing of procathepsin K to mature cathepsin K occurs as the osteoclast approaches bone, suggesting that local factors may regulate this process.     

Cathepsin K knockout mice develop osteopetrosis due to a deficit in matrix degradation but not demineralization.

Cathepsin K is a cysteine protease expressed predominantly in osteoclasts. Activated cathepsin K cleaves key bone matrix proteins and is believed to play an important role in degrading the organic phase of bone during bone resorption. Mutations in the human cathepsin K gene have been demonstrated to be associated with a rare skeletal dysplasia, pycnodysostosis. The degree of functional activity of the mutated forms of cathepsin K in these individuals has not been elucidated, but is predicted to be low or absent. To study the role of cathepsin K in bone resorption, we have generated mice deficient in the cathepsin K gene. Histologic and radiographic analysis of the mice revealed osteopetrosis of the long bones and vertebrae, and abnormal joint morphology. X-ray microcomputerized tomography images allowed quantitation of the increase in bone volume, trabecular thickness, and trabecular number in both the primary spongiosa and the metaphysis of the proximal tibiae. Not all bones were similarly affected. Chondrocyte differentiation was normal. The mice also had abnormalities in hematopoietic compartments, particularly decreased bone marrow cellularity and splenomegaly. The heterozygous animals appeared normal. Close histologic examination of bone histology revealed fully differentiated osteoclasts apposed to small regions of demineralized bone. This strongly suggests that cathepsin K-deficient osteoclasts are capable of demineralizing the extracellular matrix but are unable to adequately remove the demineralized bone. This is entirely consistent with the proposed function of cathepsin K as a matrix-degrading proteinase in bone resorption.     

Human osteoblasts produce cathepsin K

Healthy bone is a rigid yet living tissue that undergoes continuous remodeling. Osteoclasts resorb bone in the remodeling cycle. They secrete H⁺-ions and proteinases to dissolve bone mineral and degrade organic bone matrix, respectively. One of the main collagenolytic proteinase in osteoclasts is cathepsin K, a member of papain family cysteine proteinases. Recently, it has been shown that osteoblasts may contribute to organic matrix remodeling. We therefore investigated their ability to produce cathepsin K for this action. Trabecular bone samples were collected from patients operated due to a fracture of the femoral neck. Part of the bone was decalcified and the rest was used for cell isolation. Sections from the decalcified bone were immunostained with antibodies against cathepsin K. Isolated cells were characterized for their ability to form mineralized matrix and subsequently analyzed for their cathepsin K production by Western blotting and quantitative RT-PCR. Osteoblasts, bone lining cells and some osteocytes in situ showed cathepsin K immunoreactivity and osteoblast-like cells in vitro produced cathepsin K mRNA and released both 42 kDa pro- and 27 kDa processed cathepsin K to culture media. Osteoblastic cathepsin K may thus contribute to collagenous matrix maintenance and recycling of improperly processed collagen I. Whether osteoblastic cathepsin K synthesis has consequences in diseases characterized by abnormal bone matrix turnover remains to be investigated.     

Localization of Cathepsin K in Bovine Odontoclasts during Deciduous Tooth Resorption

Cathepsin K is a cysteine proteinase, which is abundantly and selectively expressed in osteoclasts. It is believed to play an important role in the proteolysis of bone resorption by osteoclasts. The objectives of this study were to investigate the association of cathepsin K in the physiological root resorption of deciduous teeth and to identify the cathepsin K-producing cells in deciduous root resorption. RT-PCR and Northern blot analysis of the total RNAs extracted from bovine active and resting root-resorbing tissues, which lie between the root of deciduous tooth and its permanent successor, were performed. The active root-resorbing tissue, which has a high population of odontoclasts on its surface that is attached to resorbing root surface, showed an extremely high expression of cathepsin K in comparison with the resting root-resorbing tissue. By in situ hybridization, cathepsin K mRNA was highly and selectively expressed in multinucleated odontoclasts that aligned along the surface of the tissue and apposed to the resorbing root surface of the deciduous tooth. Western blot analysis of the active root-resorbing tissue was used to characterize the anti-cathepsin K antibody. A band of 27 kDa, corresponding with the predicted size for mature cathepsin K, was demonstrated. Immunohistochemistry confirmed the specific localization of cathepsin K protein to the odontoclasts. These results demonstrate that odontoclasts in the deciduous root resorption express cathepsin K mRNA and protein that may participate in the proteolysis of root resorption of the deciduous tooth.     

Impaired bone resorption in cathepsin K-deficient mice is partially compensated for by enhanced osteoclastogenesis and increased expression of other proteases via an increased RANKL/OPG ratio

Previous reports indicate that mice deficient for cathepsin K (Ctsk), a key protease in osteoclastic bone resorption, develop osteopetrosis due to their inability to properly degrade organic bone matrix. Some features of the phenotype of Ctsk knockout mice, however, suggest the presence of mechanisms by which Ctsk-deficient mice compensate for the lack of cathepsin K. To study these mechanisms in detail, we generated Ctsk-deficient (Ctsk-/-) mice and analyzed them at the age of 2, 7, and 12 months using peripheral quantitative computed tomography, histomorphometry, resorption marker measurements, osteoclast and osteoblast differentiation cultures, and gene expression analyses. The present study verified the previously published osteopetrotic features of Ctsk-deficient mice. However, these changes did not exacerbate during aging indicating the absence of Ctsk to have its most severe effects during the rapid growth period. Resorption markers ICTP and CTX were decreased in the media of Ctsk-/- osteoclasts cultured on bone slices indicating impaired bone resorption. Ctsk-/- mice exhibited several mechanisms attempting to compensate for Ctsk deficiency. The number of osteoclasts in trabecular bone was significantly increased in Ctsk-/- mice compared to controls, as was the number of osteoclast precursors in bone marrow. The mRNA levels for receptor activator of nuclear factor (kappa)B ligand (RANKL) in Ctsk-/- bones were increased resulting in increased RANKL/OPG ratio favoring osteoclastogenesis. In addition, expression of mRNAs of osteoclastic enzymes (MMP-9, TRACP) and for osteoblastic proteases (MMP-13, MMP-14) were increased in Ctsk-/- mice compared to controls. Impaired osteoclastic bone resorption in Ctsk-/- mice results in activation of osteoblastic cells to produce increased amounts of other proteolytic enzymes and RANKL in vivo. We suggest that increased RANKL expression mediates enhanced osteoclastogenesis and increased protease expression by osteoclasts. These observations underline the important role of osteoblastic cells in regulation of osteoclast activity and bone turnover.     

Conditioned medium from ras oncogene-transformed NIH 3T3 cells induces bone resorption in vitro

Tumor-associated hypercalcemia is due, in part, to enhanced osteoclastic bone resorption induced by soluble factors elaborated from malignant cells. ras transformation of NIH 3T3 cells results in a 50-fold induction of cathepsin L mRNA and secretion of the corresponding protein. Since cathepsin L is an acid proteinase we asked whether conditioned medium from these cells would directly increase calcium release from bone in vitro. We tested conditioned medium obtained after 72 h culture of NIH 3T3 ras-transformed cells (DT) or nontransformed NIH 3T3 cells (3T3) and identical medium not exposed to cells (Ctl). Incubation of either live or dead neonatal mouse calvaria for 48 h in DT-conditioned medium increased calcium release compared to bones incubated with 3T3 medium. In both states the increased calcium release with DT medium was blocked by 0.25 mM E-64, a general cysteine proteinase inhibitor, and 1 microM Z-Phe-Ala-CH2F, a specific inhibitor of cathepsin L activity. Thus, conditioned medium from ras-transformed cells enhances calcium release in both live and dead bone. Since cathepsin L is the major protein secreted by these cells and the effect of DT-conditioned medium is blocked by a specific inhibitor of cathepsin L, these studies suggest that this acid proteinase acts directly on bone mineral to enhance net calcium release.     

Degradation of the organic phase of bone by osteoclasts: a secondary role for lysosomal acidification.

Osteoclasts degrade bone matrix by secretion of hydrochloric acid and proteases. We studied the processes involved in the degradation of the organic matrix of bone in detail and found that lysosomal acidification is involved in this process and that MMPs are capable of degrading the organic matrix in the absence of cathepsin K. INTRODUCTION: Osteoclasts resorb bone by secretion of acid by the vacuolar H+-adenosine triphosphatase (V-ATPase) and the chloride channel ClC-7, followed by degradation of the matrix, mainly collagen type I, by cathepsin K and possibly by matrix metalloproteinases (MMPs). However, the switch from acidification to proteolysis and the exact roles of both the ion transporters and the proteinases still remain to be studied. MATERIALS AND METHODS: We isolated CD14+ monocytes from human peripheral blood from either controls or patients with autosomal dominant osteopetrosis type II (ADOII) caused by defective ClC-7 function and cultured them in the presence of RANKL and macrophage-colony stimulating factor (M-CSF) to generate osteoclasts. We decalcified cortical bovine bone slices and studied the osteoclasts with respect to morphology, markers, and degradation of the decalcified matrix in the presence of various inhibitors of osteoclast acidification and proteolysis, using normal calcified bone as a reference. RESULTS: We found that ADOII osteoclasts not only have reduced resorption of the calcified matrix, but also 40% reduced degradation of the organic phase of bone. We found that both acidification inhibitors and cathepsin K inhibitors reduced degradation of the organic matrix by 40% in normal osteoclasts, but had no effect in the ADOII osteoclasts. Furthermore, we showed that inhibition of MMPs leads to a 70% reduction in the degradation of the organic bone matrix and that MMPs and cathepsin K have additive effects. Finally, we show that osteoclastic MMPs mediate release of the carboxyterminal telopeptide of type I collagen (ICTP) fragment in the absence of cathepsin K activity, and therefore, to some extent, are able to compensate for the loss of cathepsin K activity. CONCLUSIONS: These data clearly show that osteoclastic acidification of the lysosomes plays a hitherto nonrecognized role in degradation of the organic matrix. Furthermore, these data shed light on the complicated interplay between acidification dependent and independent proteolytic processes, mediated by cathepsin K and the MMPs, respectively.     

The relative contribution of cysteine proteinases and matrix metalloproteinases to the resorption process in osteoclasts derived from long bone and scapula

It has been suggested that functional heterogeneity exists between osteoclasts from different bone sites. This could be exploited to design therapeutics that would selectively inhibit bone resorption only at compromised sites. To further investigate the existence of functional differences between osteoclasts from different bone sites we assessed whether osteoclasts isolated from intramembranous bone differ from osteoclasts isolated from endochondral bone in the extent that they utilize cysteine proteinases and matrix metalloproteinases to degrade the organic matrix of bone. The differential involvement of the two classes of proteases was assessed by analyzing dose-dependent effects of the matrix metalloproteinase inhibitor, CT-1746, and of the cathepsin inhibitor, E64, on bone resorption. Osteoclasts isolated from the scapula (intramembranous) and long bones (endochondral) of newborn New Zealand white rabbits were seeded on cortical bovine bone slices in the presence or absence of inhibitors. Resorptive activity was evaluated by measuring the number and area of resorption pits and by measuring the release of collagen degradation products in the culture medium. In the absence of inhibitors, scapular osteoclasts and long bone osteoclasts had similar activity based on these criteria. The resorptive activity of scapular osteoclasts was inhibited to a greater extent by the MMP inhibitor CT-1746 than by the cysteine proteinase inhibitor E64. Conversely, resorption by osteoclasts derived from long bones was inhibited to a greater degree by the cysteine proteinase inhibitor. These results strongly suggest that there are functional differences between dispersed osteoclasts derived from the scapula and long bones, with scapular osteoclasts utilizing matrix metalloproteinases to a greater extent than cysteine proteinases and long bone osteoclasts using cysteine proteinases to a greater extent than matrix metalloproteinases.     

Effects of antisense mediated inhibition of cathepsin K on human osteoclasts obtained from peripheral blood.

Cathepsin K is a cystein protease that displays a proteolytic activity against Type I collagen and is abundantly and selectively expressed in osteoclasts where it plays a critical role in bone degradation. Its direct role in bone tissue has been defined by knock-out mice studies and inhibiting strategies in animals models. However, direct proof of cathepsin K function in human osteoclast model in vitro is lacking. The aim of this study is to analyze cathepsin K expression and localization in human osteoclasts obtained from peripheral blood and to examine cathepsin K function in these cells by antisense oligodeoxynucleotide (AS-ODN) strategy. AS-ODN was added to the culture of osteoclast precursors induced to differentiate by RANKL and M-CSF. AS-ODN treatment produced a significant down-regulation of cathepsin K mRNA (>80%) and protein expression, as verified respectively by Real-time PCR and by immunocytochemistry or Western blot. The cathepsin K inhibition caused an impairment of resorption activity as evaluated by a pit formation assay ( p = 0.045) and by electron microscopy, while the acidification process was unaffected. We demonstrated that antisense strategies against cathepsin K are selectively effective to inhibit resorption activity in human osteoclasts, like in animal models.     

Development and characterization of a human in vitro resorption assay: demonstration of utility using novel antiresorptive agents.

A human in vitro resorption assay has been developed using osteoclastoma-derived osteoclasts and used to evaluate novel antiresorptive agents including antagonists of the alphavbeta3 integrin, and inhibitors of cathepsin K and the osteoclast ATPase. The potency of novel compounds in the in vitro resorption assay correlates with functional assays for each class of inhibitor: the human alphavbeta3-mediated cell adhesion assay for the vitronectin receptor antagonists (r2 = 0.82), the chick osteoclast vacuolar ATPase enzyme assay for the H+-ATPase inhibitors (r2 = 0.77) and the recombinant human cathepsin K enzyme assay for the cathepsin K inhibitors (r2 = 0.80). Cell suspensions, rich in osteoclasts, are prepared by collagenase digestion of the tumor tissue. These cells can be stored long-term in liquid nitrogen and upon thawing maintain their bone-resorbing phenotype. The cryopreserved cells can be cultured on bovine cortical bone for 24-48 h and resorption can be measured by either confocal microscopy or biochemical assays. The resorptive activity of osteoclasts derived from a number of tumors can be inhibited reproducibly using a number of mechanistically unique antiresorptive compounds. In addition, the measurement of resorption pits by laser confocal microscopy correlates with the release of type I collagen C-telopeptides or N-telopeptides, as measured by enzyme-linked immunosorbent assay. Resorption can be measured reproducibly using a 48-h incubation of osteoclasts on bone slices, or a 24-h incubation with bone particles. This in vitro human osteoclast resorption assay provides a robust system for the evaluation of inhibitors of osteoclastic function that may be developed for the treatment of metabolic bone diseases such as osteoporosis.     

Ablation of Cathepsin K Activity in the Young Mouse Causes Hypermineralization of Long Bone and Growth Plates

Cathepsin K deficiency in humans causes pycnodysostosis, which is characterized by dwarfism and osteosclerosis. Earlier studies of 10-week-old male cathepsin K-deficient (knockout, KO) mice showed their bones were mechanically more brittle, while histomorphometry showed that both osteoclasts and osteoblasts had impaired activity relative to the wild type (WT). Here, we report detailed mineral and matrix analyses of the tibia of these animals based on Fourier transform infrared microspectroscopy and imaging. At 10 weeks, there was significant hypercalcification of the calcified cartilage and cortices in the KO. Carbonate content was elevated in the KO calcified cartilage as well as cortical and cancellous bone areas. These data suggest that cathepsin K does not affect mineral deposition but has a significant effect on mineralized tissue remodeling. Since growth plate abnormalities were extensive despite reported low levels of cathepsin K expression in the calcified cartilage, we used a differentiating chick limb-bud mesenchymal cell system that mimics endochondral ossification but does not contain osteoclasts, to show that cathepsin K inhibition during initial stages of mineral deposition retards the mineralization process while general inhibition of cathepsins can increase mineralization. These data suggest that the hypercalcification of the cathepsin K-deficient growth plate is due to persistence of calcified cartilage and point to a role of cathepsin K in bone tissue development as well as skeletal remodeling.     

A potent small molecule, nonpeptide inhibitor of cathepsin K (SB 331750) prevents bone matrix resorption in the ovariectomized rat

Inhibition of the cyteine proteinase, cathepsin K (E.C. 3.4.22.38) has been postulated as a means to control osteoclast-mediated bone resorption. The preferred animal models for evaluation of antiresorptive activity are in the rat. However, the development of compounds that inhibit rat cathepsin K has proven difficult because the human and rat enzymes differ in key residues in the active site. In this study, a potent, nonpeptide inhibitor of rat cathepsin K (K(i) = 4.7 nmol/L), 5-(2-morpholin-4-yl-ethoxy)-benzofuran-2-carboxylic acid ((S)-3-methyl-1-(3-oxo-1-[2-(3-pyridin-2-yl-phenyl)-ethenoyl]-azepan-4-ylcarbanoyl)-butyl)-amide (SB 331750), is described, which is efficacious in rat models of bone resorption. SB 331750 potently inhibited human cathepsin K activity in vitro (K(i) = 0.0048 nmol/L) and was selective for human cathepsin K vs. cathepsins B (K(i) = 100 nmol/L), L (0.48 nmol/L), or S (K(i) = 14.3 nmol/L). In an in situ enzyme assay, SB 331750 inhibited osteoclast-associated cathepsin activity in tissue sections containing human osteoclasts (IC(50) approximately 60 nmol/L) and this translated into potent inhibition of human osteoclast-mediated bone resorption in vitro (IC(50) approximately 30 nmol/L). In vitro, SB 331750 partially, but dose-dependently, prevented the parathyroid hormone-induced hypercalcemia in an acute rat model of bone resorption. To evaluate the ability of SB 331750 to inhibit bone matrix degradation in vivo, it was administered for 4 weeks at 3, 10, or 30 mg/kg, intraperitoneally (i.p.), u.i.d. in the ovariectomized (ovx) rat. Both 10 and 30 mg/kg doses of compound prevented the ovx-induced elevation in urinary deoxypyridinoline and prevented the ovx-induced increase in percent eroded perimeter. Histological evaluation of the bones from compound-treated animals indicated that SB 331750 retarded bone matrix degradation in vivo at all three doses. The inhibition of bone resorption at the 10 and 30 mg/kg doses resulted in prevention of the ovx-induced reduction in percent trabecular area, trabecular number, and increase in trabecular spacing. These effects on bone resorption were also reflected in inhibition of the ovx-induced loss in trabecular bone volume as assessed using microcomputerized tomography (microCT; approximately 60% at 30 mg/kg). Together, these data indicate that the cathepsin K inhibitor, SB 331750, prevented bone resorption in vivo and this inhibition resulted in prevention of ovariectomy-induced loss in trabecular structure.