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.     

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 .     

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.     

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.     

The bone lining cell: its role in cleaning Howship's lacunae and initiating bone formation.

In this study we investigated the role of bone lining cells in the coordination of bone resorption and formation. Ultrastructural analysis of mouse long bones and calvariae revealed that bone lining cells enwrap and subsequently digest collagen fibrils protruding from Howship's lacunae that are left by osteoclasts. By using selective proteinase inhibitors we show that this digestion depends on matrix metalloproteinases and, to some extent, on serine proteinases. Autoradiography revealed that after the bone lining cells have finished cleaning, they deposit a thin layer of a collagenous matrix along the Howship's lacuna, in close association with an osteopontin-rich cement line. Collagenous matrix deposition was detected only in completely cleaned pits. In bone from pycnodysostotic patients and cathepsin K-deficient mice, conditions in which osteoclastic bone matrix digestion is greatly inhibited, bone matrix leftovers proved to be degraded by bone lining cells, thus indicating that the bone lining cell "rescues" bone remodeling in these anomalies. We conclude that removal of bone collagen left by osteoclasts in Howship's lacunae is an obligatory step in the link between bone resorption and formation, and that bone lining cells and matrix metalloproteinases are essential in this process.     

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.     

Proteolysis of human bone collagen by cathepsin K: characterization of the cleavage sites generating by cross-linked N-telopeptide neoepitope

An immunoassay for cross-linked N-telopeptides of type I collagen (NTx) in urine or serum has proven to give a sensitive index of osteoclast-mediated bone resorption. We show that recombinant human cathepsin K is highly active in releasing the NTx neoepitope in 100% yield from bone type I collagen. Cathepsins S, L, and B were also active but at 57%, 36%, and 27% of the yield of K, respectively. The matrix metalloproteinases that were tested, stromelysin, collagenase 3, or matrilysin, did not produce any immunoreactivity. Cathepsin K also acted on demineralized bone matrix, releasing NTx epitope and completely dissolving the bone particles in 24-48 h. Proteolytic cleavage of a G-L peptide bond in the alpha2(I)N-telopeptide was shown to be required for recognition by monoclonal antibody 1H11. Peptide analysis identified bonds in the N-telopeptide and helical cross-linking domains adjacent to the cross-linking residues at which cathepsin K cleaved in bone collagen. The sites were consistent with the known substrate specificity of cathepsin K, which prefers a hydrophobic residue or proline in the critical P2 position. The NTx peptides generated by cathepsin K were of low molecular weight, in the range previously found in human urine. Because cathepsin K appears to be essential for the normal resorption of mineralized bone matrix by osteoclasts, these findings help explain the specificity and responsiveness of NTx as a marker of osteoclastic bone resorption in vivo.     

Matrix metalloproteinase-12 (MMP-12) in osteoclasts: new lesson on the involvement of MMPs in bone resorption

Osteoclasts require matrix metalloproteinase (MMP) activity and cathepsin K to resorb bone, but the critical MMP has not been identified. Osteoclasts express MMP-9 and MMP-14, which do not appear limiting for resorption, and the expression of additional MMPs is not clear. MMP-12, also called metalloelastase, is reported only in a few cells, including tissue macrophages and hypertrophic chondrocytes. MMP-12 is critical for invasion and destruction in pathologies such as aneurysm and emphysema. In the present study, we demonstrate that osteoclasts express MMP-12, although only in some situations. Northern blots show that highly purified rabbit osteoclasts in culture express MMP-12 at the same level as macrophages, whereas in situ hybridizations performed on rabbit bone do not show any MMP-12 expression in osteoclasts whatever the bone type. In contrast, in situ hybridizations performed on mouse bone show MMP-12 expression in osteoclasts in calvariae and long bones. We also demonstrate that recombinant MMP-12 cleaves the putative functional domains of osteopontin and bone sialoprotein, two bone matrix proteins that strongly influence osteoclast activities, such as attachment, spreading and resorption. Furthermore, we investigated the role of MMP-12 in bone resorption and osteoclast recruitment by comparing MMP-12 knockout and wild-type mice in specialized culture models known to depend on MMP activity, as well as in the ovariectomy model, and we did not find any indication for a limiting role of MMP-12 in these processes. In conclusion, we found that osteoclasts are able to express MMP-12, but MMP-12 did not appear critical for osteoclast recruitment or resorption. The fact that none of the MMPs identified so far in osteoclasts appears limiting for resorption, gives strength to the hypothesis that the critical MMP for bone solubilization is produced by non-osteoclastic cells.     

Cathepsin K: its skeletal actions and role as a therapeutic target in osteoporosis

Bone remodeling consists of two phases--bone resorption and bone formation--that are normally balanced. When bone resorption exceeds bone formation, pathologic processes, such as osteoporosis, can result. Cathepsin K is a member of the papain family of cysteine proteases that is highly expressed by activated osteoclasts. Cathepsin K readily degrades type I collagen, the major component of the organic bone matrix. With such a major role in the initial process of bone resorption, cathepsin K has become a therapeutic target in osteoporosis. The antiresorptive properties of cathepsin K inhibitors have been studied in phase I and phase II clinical trials. Phase III studies are currently underway for odanacatib, a selective cathepsin K inhibitor.     

Inhibition of cathepsin K for treatment of osteoporosis

Cathepsin K is the protease that is primarily responsible for the degradation of bone matrix by osteoclasts. Inhibitors of cathepsin K are in development for treatment of osteoporosis. Currently available antiresorptive drugs interfere with osteoclast function. They inhibit both bone resorption and formation, due to the coupling between these processes. Cathepsin K inhibitors, conversely, target the resorption process itself and may not interfere with osteoclast stimulation of bone formation. In fact, when cathepsin K is absent or inhibited in mice, rabbits, or monkeys, bone formation is maintained or increased. In humans, inhibition of cathepsin K is associated with sustained reductions in bone resorption markers but with smaller and transient reductions in bone formation markers. The usefulness of cathepsin K inhibitors in osteoporosis is now being examined in phase 2 and phase 3 clinical trials of postmenopausal osteoporotic women.     

Accelerated turnover of metaphyseal trabecular bone in mice overexpressing cathepsin K.

This study is based on a hypothesis that overexpression of an osteoclast enzyme, cathepsin K, causes an imbalance in bone remodeling toward bone loss. The hypothesis was tested in transgenic (TG) mice harboring additional copies of the murine cathepsin K gene (Ctsk) identifiable by a silent mutation engineered into the construct. For this study, three TG mouse lines harboring 3-25 copies of the transgene were selected. Tissue specificity of transgene expression was determined by Northern analysis, which revealed up to 6-fold increases in the levels of cathepsin K messenger RNA (mRNA) in calvarial and long bone samples of the three TG lines. No changes were seen in the mRNA levels of other osteoclast enzymes, indicating that the increase in cathepsin K mRNA was not a reflection of activation of all osteoclast enzymes. Immunohistochemistry confirmed that cathepsin K expression in the TG mice was confined to osteoclasts and chondroclasts. Histomorphometry revealed a significantly decreased trabecular bone volume (BV), but, surprisingly, also a marked increase in the number of osteoblasts, the rate of bone turnover, and the amount of mineralizing surface (MS). However, monitoring of bone density in the proximal tibias of the TG mice with peripheral quantitative computed tomography (pQCT) failed to reveal statistically significant changes in bone density. Similarly, no statistically significant alterations were observed in biomechanical testing at the age of 7 months. The increases in parameters of bone formation triggered by increased cathepsin K expression is an example of the tight coupling of bone resorption and formation during the bone-remodeling cycle.     

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.     

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.     

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.     

Histochemical examination of osteoblastic activity in op/op mice with or without injection of recombinant M-CSF

Osteopetrotic (op/op) mice do not exhibit bone remodeling because of defective osteoclast formation caused by the depletion of macrophage colony-stimulating factor (M-CSF). In the present study, we investigated tibial bones of op/op mice with or without prior injections of M-CSF to determine whether osteoclast formation and subsequent bone resorption could activate osteoblasts, which is known as a "coupling" phenomenon. In op/op mice, no osteoclasts were present, but the metaphyseal osteoblasts adjacent to the growth plate cartilage seemed to be active, revealing an intense alkaline phosphatase (ALPase) immunoreactivity. Consequently, primary trabecular bones were extended continuously to the diaphysis, indicating that bone modeling is well achieved in op/op mice. In contrast with the metaphysis, most of the diaphyseal osteoblasts were flattened and showed weak ALPase activity, and, as a result, they seemed to be less active. Osteopontin (OPN) was localized slightly at the interface between bone and cartilage matrices of the primary trabeculae. In contrast, in op/op mice injected with M-CSF, tartrate-resistant acid phosphatase-positive osteoclasts appeared, resorbing trabecular bones of the diaphyseal region. The diaphyseal osteoblasts in the vicinity of the active osteoclasts were cuboidal and exhibited strong ALPase immunoreactivity. OPN was observed not only at the bone-cartilage interface, but also significantly on the resorption lacunae beneath the bone-resorbing osteoclasts. These observations indicate that the activation of diaphyseal osteoblasts appears to be coupled with osteoclast formation and subsequent osteoclastic bone resorption. Alternatively, the metaphyseal osteoblasts at the chondro-osseous junction seemed to be less affected by osteoclastic activity.     

Family 2 cystatins inhibit osteoclast-mediated bone resorption in calvarial bone explants

Osteoclastic bone resorption depends on the activity of various proteolytic enzymes, in particular those belonging to the group of cysteine proteinases. Biochemical studies have shown that cystatins, naturally occurring inhibitors of these enzymes, inhibit bone matrix degradation. Since the mechanism by which cystatins exert this inhibitory effect is not completely resolved yet, we studied the effect of cystatins on bone resorption microscopically and by Ca-release measurements. Calvarial bone explants were cultured in the presence or absence of family 2 cystatins and processed for light and electron microscopic analysis, and the culture media were analyzed for calcium release. Both egg white cystatin and human cystatin C decreased calcium release into the medium significantly. Microscopic analyses of the bone explants demonstrated that in the presence of either inhibitor, a high percentage of osteoclasts was associated with demineralized non-degraded bone matrix. Following a 24-h incubation in the presence of cystatin C, 41% of the cells were adjacent to areas of demineralized non-degraded bone matrix, whereas in controls, this was only 6%. If bone explants were cultured with both PTH and cystatin C, 60% of the osteoclasts were associated with demineralized non-degraded bone matrix, compared to 27% for bones treated with PTH only (P < 0.01). Our study provides evidence that cystatins, the naturally occurring inhibitors of cysteine proteinases, reversibly inhibit bone matrix degradation in the resorption lacunae adjacent to osteoclasts. These findings suggest the involvement of cystatins in the modulation of osteoclastic bone degradation.     

Differential turnover of cortical and trabecular bone in transgenic mice overexpressing cathepsin K

Cathepsin K is a major osteoclastic protease. We have recently shown that overexpression of mouse cathepsin K gene in transgenic UTU17 mouse model results in high turnover osteopenia of metaphyseal trabecular bone at the age of 7 months. The present report extends these studies to a systematic analysis of cortical bone in growing and adult mice overexpressing cathepsin K. Mice homozygous for the transgene locus (UTU17+/+) and their control littermates were studied at the age of 1, 3, 7, and 12 months. Bone properties were analyzed using peripheral quantitative computed tomography (pQCT), histomorphometry, histochemistry, radiography, and biomechanical testing. In addition, the levels of biochemical markers of bone turnover were measured in the sera. Unexpectedly, cortical thickness and cortical bone mineral density were increased in the diaphyseal region of growing and adult UTU17+/+ mice. This was associated with an increased number of vascular canals leading to increased cortical porosity in UTU17+/+ mice without changes in the ultimate bending force or stiffness of the bone. In UTU17+/+ mice, osteopenia of metaphyseal trabecular bone was observed already at the age of 1 month. In sera of 1-month-old UTU17+/+ mice, the activity of tartrate-resistant acid phosphatase 5b was decreased and the levels of osteocalcin increased. Our results support the role of cathepsin K as a major proteinase in osteoclastic bone resorption. Excessive production of cathepsin K induced osteopenia of metaphyseal trabecular bone and increased the porosity of diaphyseal cortical bone. The increased cortical thickness and bone mineral density observed in diaphyses of UTU17+/+ mice demonstrate the different nature and reactivity of trabecular and cortical bone in mice. These results suggest that the biomechanical properties of cortical bone are preserved through adaptation as outlined in Wolff's law.     

Implications of Osteoblast-Osteoclast Interactions in the Management of Osteoporosis by Antiresorptive Agents Denosumab and Odanacatib

Antiresorptive agents, used in the treatment of osteoporosis, inhibit either osteoclast formation or function. However, with these approaches, osteoblast activity is also reduced because of the loss of osteoclast-derived coupling factors that serve to stimulate bone formation. This review discusses how osteoclast inhibition influences osteoblast function, comparing the actions of an inhibitor of osteoclast formation [anti-RANKL/Denosumab (DMAB)] with that of a specific inhibitor of osteoclastic cathepsin K activity [Odanacatib (ODN)]. Denosumab rapidly and profoundly, but reversibly, reduces bone formation. In contrast, preclinical studies and clinical trials of ODN showed that bone formation at some skeletal sites was preserved although resorption was reduced. This preservation of bone formation appears to be due to effects of coupling factors, secreted by osteoclasts and released from demineralized bone matrix. This indicates that bone resorptive activities of osteoclasts are separable from their coupling activities.     

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.     

Potent and selective inhibition of human cathepsin K leads to inhibition of bone resorption in vivo in a nonhuman primate.

Cathepsin K is a cysteine protease that plays an essential role in osteoclast-mediated degradation of the organic matrix of bone. Knockout of the enzyme in mice, as well as lack of functional enzyme in the human condition pycnodysostosis, results in osteopetrosis. These results suggests that inhibition of the human enzyme may provide protection from bone loss in states of elevated bone turnover, such as postmenopausal osteoporosis. To test this theory, we have produced a small molecule inhibitor of human cathepsin K, SB-357114, that potently and selectively inhibits this enzyme (Ki = 0.16 nM). This compound potently inhibited cathepsin activity in situ, in human osteoclasts (inhibitor concentration [IC]50 = 70 nM) as well as bone resorption mediated by human osteoclasts in vitro (IC50 = 29 nM). Using SB-357114, we evaluated the effect of inhibition of cathepsin K on bone resorption in vivo using a nonhuman primate model of postmenopausal bone loss in which the active form of cathepsin K is identical to the human orthologue. A gonadotropin-releasing hormone agonist (GnRHa) was used to render cynomolgus monkeys estrogen deficient, which led to an increase in bone turnover. Treatment with SB-357114 (12 mg/kg subcutaneously) resulted in a significant reduction in serum markers of bone resorption relative to untreated controls. The effect was observed 1.5 h after the first dose and was maintained for 24 h. After 5 days of dosing, the reductions in N-terminal telopeptides (NTx) and C-terminal telopeptides (CTx) of type I collagen were 61% and 67%, respectively. A decrease in serum osteocalcin of 22% was also observed. These data show that inhibition of cathepsin K results in a significant reduction of bone resorption in vivo and provide further evidence that this may be a viable approach to the treatment of postmenopausal osteoporosis.