Study of immunoelectron microscopic localization of cathepsin K in osteoclasts and other bone cells in the mouse femur

The localization of cathepsin K protein in mouse osteoclasts was examined by immunolight and immunoelectron microscopy using the avidin-biotin-peroxidase complex method with anti-cathepsin K (mouse) antibody. With light microscopy, a strong immunoreaction for cathepsin K was found extracellularly along the bone and cartilage resorption lacunae and detected intracellularly in vesicles, granules, and vacuoles throughout the cytoplasm of multinuclear osteoclasts and chondroclasts attached to the surface of the bone or cartilage. Mononuclear cells, probably preosteoclasts, some distance from the bone also contained a few cathepsin K-positive vesicles and granules. Cathepsin K was sometimes found in the cisternal spaces of the rough endoplasmic reticulum and vesicles of the Golgi apparatus with electron microscopy of the basolateral region of the osteoclasts. Cathepsin K-positive vesicles and granules as lysosomal compartments were present in various stages of fusion with vacuoles as endosomal compartments that contained fragmented cathepsin K-negative fibril-like structures. Some of the vacuoles (endolysosomes), which seemed to be formed by this process of fusion, contained cathepsin K-positive vesicles and fibril-like structures that did not show the regular cross striation of type I collagen fibrils. In the apical region of the osteoclasts, cathepsin K-positive vesicles and pits had already fused with or were in the process of fusing with the ampullar extracellular spaces. There were large deposits of cathepsin K on fragmented fibril-like structures without regular cross striation in the extracellular spaces, as well as on and between the cytoplasmic processes of the ruffled border. There were also extensive deposits of cathepsin K on the type I collagen fibrils with cross striation in the bone resorption lacunae. Osteoblasts and osteocytes were negative for cathepsin K. In the immunocytochemical controls, no immunoreaction was found in the osteoclasts or preosteoclasts, or on the collagen fibrils in the resorption lacunae. The results indicate that cathepsin K is produced in mature osteoclasts attached to the bone and secreted into the bone resorption lacunae. The findings suggest that cathepsin K participates in the extracellular degradation of collagen fibrils in the resorption lacunae and in the subsequent degradation of the fragmented fibrils in the endolysosomes. It is also suggested that cathepsin K degrades the organic cartilage matrix.     

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.     

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.     

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.     

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.     

Protein kinase C–delta deficiency perturbs bone homeostasis by selective uncoupling of cathepsin K secretion and ruffled border formation in osteoclasts

Bone homeostasis requires stringent regulation of osteoclasts, which secrete proteolytic enzymes to degrade the bone matrix. Despite recent progress in understanding how bone resorption occurs, the mechanisms regulating osteoclast secretion, and in particular the trafficking route of cathepsin K vesicles, remain elusive. Using a genetic approach, we describe the requirement for protein kinase C–delta (PKCδ) in regulating bone resorption by affecting cathepsin K exocytosis. Importantly, PKCδ deficiency does not perturb formation of the ruffled border or trafficking of lysosomal vesicles containing the vacuolar‐ATPase (v‐ATPase). Mechanistically, we find that cathepsin K exocytosis is controlled by PKCδ through modulation of the actin bundling protein myristoylated alanine‐rich C‐kinase substrate (MARCKS). The relevance of our finding is emphasized in vivo because PKCδ?/? mice exhibit increased bone mass and are protected from pathological bone loss in a model of experimental postmenopausal osteoporosis. Collectively, our data provide novel mechanistic insights into the pathways that selectively promote secretion of cathepsin K lysosomes independently of ruffled border formation, providing evidence of the presence of multiple mechanisms that regulate lysosomal exocytosis in osteoclasts. © 2012 American Society for Bone and Mineral Research.     

Cytological and functional studies of preosteoclasts and osteoclasts in the alveolar bones from neonatal rats using microperoxidase as a tracer

Summary Microperoxidase (MP) was used to investigate the cytological and functional features of preosteoclasts and osteoclasts during rat alveolar bone development. We observed mononuclear cells as preosteoclasts and multinuclear cells with and without ruffled borders (RB). In the bone facing multinuclear cells with RB as active osteoclasts, MP was extensively deposited along the external bone matrix undergoing resorption, and was phagocytosed with bone components into the vacuoles of osteoclasts. Neither preosteoclasts nor multinuclear cells without RB took up MP and bone components. Only multinuclear cells with RB seemed to resorb bone. Monocytes/macrophages (MMP) phagocytosed MP through all regions of the plasma membrane, whereas osteoclasts took up MP only through the RB which was a part of the plasma membrane. Endogenous peroxidase was detected in the MMP but not in preosteoclasts and osteoclasts. Thus, osteoclasts were considerably different from the MMP. The numbers of MMP were extremely few close to the osteoclasts, whereas moderate numbers of preosteoclasts were located close to the osteoclasts. Except for the nucleus and RB, there were many morphological similarities between preosteoclasts and osteoclasts. We therefore suggest that preosteoclasts, rather than MMP, are the precursors of osteoclasts during alveolar bone development of neonatal rats.     

Immunolocalization of the cation-independent mannose 6-phosphate receptor and cathepsin B in the enamel organ and alveolar bone of the rat incisor

In order to examine our hypothesis that maturation ameloblasts could degrade the enamel matrix in a manner analogous to bone resorption mediated by osteoclasts, we have assessed the distribution of lysosomal enzymes in the enamel organ by immunolocalizing the cation-inindependent mannose 6-phosphate receptor (MPR) and the lysosomal enzyme cathepsin B at all stages of amelogenesis. Secretory ameloblasts showed strong immunoreactivity for MPR in the supranuclear Golgi region and in the cytoplasm between the Golgi region and the distal junctional complexes. However, cathepsin B immunoreactivity was mainly seen in the distal portion of Tomes' process, which was unreactive for MPR immunogenicity. In maturation ameloblasts, the MPR was observed on the ruffled border of the ruffle-ended ameloblast (RA) but not on the distal cell membrane of the smooth-ended ameloblast (SA), although both cell types demonstrated strong immunoreactivity for MPR in the Golgi region. Immunoreactive cathepsin B was seen at the distal ends of both RA and SA. It is postulated that the nascent lysosomal enzymes bind to the mannose 6-phosphate receptors which target them not only to intracellular lysosomes, but also to the ruffled border of maturation ameloblasts where these enzymes are secreted into the enamel. Since MPR and lysosomal enzymes were also detected on the ruffled border of osteoclasts (Ocl) adjacent to alveolar bone, our immunocytochemical approach provides strong evidence for a similarity between the maturation process in enamel, as mediated by the ruffle-ended maturation ameloblasts, and bone resorption mediated by osteoclasts. This study has established that a common mechanism, based on MPR-targeted lysosomal secretion and matrix degradation, is basic to the maturation process involved in calcified tissues as different as bone and enamel.     

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.     

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.     

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.     

Phenytoin-Induced Bone Loss and Its Prevention with Alfacalcidol or Calcitriol in Growing Rats

Cathepsin K is a lysosomal cysteine proteinase (LCP) predominantly expressed in osteoclasts. This study was conducted to evaluate the improtance of human cathepsin K for osteoclastic bone resorption relative to that of other LCPs. To accomplish this, we quantitatively determined the expression levels of major LCPs (cathepsins B, K, L, and S) in human osteoclastic cells by using competitive RT-PCR. Giant cell tumor of bone (GCT) was used as a source of human osteoclastic cells, since the tissue was shown to contain a large number of cells satisfying the criteria for typical osteoclasts. The involvement of LCPs in the bone-resorption process by the GCT cells was confirmed by showing thattrans-epoxysucciny-l-leucylamido-(4-guanidino) butane (E-64), a nonselective cysteine proteinase inhibitor, exerted an inhibitory effect on the pit formation. We isolated osteoclast-like cells (OLCs) positive for tartrate-resistant acid phosphatase (TRAP) and cathepsin K from the GCT tissue to a degree of almost 95% purity. In these cells, the expression of cathepsin K was shown to be approximately 20-, 130-, and 410-fold stronger than that of cathepsins B, L, and S, respectively. A similar result was obtained when human bone marrow cells in culture were used as another source of OLCs. Further, we found that cathepsin K was expressed in OLCs far more strongly than in several human nonosteoclastic cells including osteoblastic cell lines. The abundant and selective expression of cathepsin K in OLCs relative to that of other LCPs suggests that cathepsin K is mainly responsible for osteoclastic degradation of human bone matrix.     

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 .     

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.     

V-ATPase subunit ATP6AP1 (Ac45) regulates osteoclast differentiation, extracellular acidification, lysosomal trafficking, and protease exocytosis in osteoclast-mediated bone resorption

Lysosomal trafficking and protease exocytosis in osteoclasts are essential for ruffled border formation and bone resorption. Yet the mechanism underlying lysosomal trafficking and the related process of exocytosis remains largely unknown. We found ATP6ap1 (Ac45), an accessory subunit of vacuolar-type H(+)-ATPases (V-ATPases), to be highly induced by receptor activator for nuclear factor kappa B ligand (RANKL) in osteoclast differentiation. Ac45 knockdown osteoclasts formed normal actin rings, but had severely impaired extracellular acidification and bone resorption. Ac45 knockdown significantly reduced osteoclast formation. The decrease in the number of osteoclasts does not result from abnormal apoptosis; rather, it results from decreased osteoclast precursor cell proliferation and fusion, which may be partially due to the downregulation of extracellular signal-regulated kinase (ERK) phosphorylation and FBJ osteosarcoma oncogene (c-fos), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), and "transmembrane 7 superfamily member 4" (Tm7sf4) expression. Notably, Ac45 knockdown osteoclasts exhibited impaired lysosomal trafficking and exocytosis, as indicated by the absence of lysosomal trafficking to the ruffled border and a lack of cathepsin K exocytosis into the resorption lacuna. Our data revealed that the impaired exocytosis is specifically due to Ac45 deficiency, and not the general consequence of a defective V-ATPase. Together, our results demonstrate the essential role of Ac45 in osteoclast-mediated extracellular acidification and protease exocytosis, as well as the ability of Ac45 to guide lysosomal intracellular trafficking to the ruffled border, potentially through its interaction with the small guanosine-5'-triphosphatase (GTPase) Rab7. Our work indicates that Ac45 may be a novel therapeutic target for osteolytic disease.     

Polarized osteoclasts put marks of tartrate-resistant acid phosphatase on dentin slices--a simple method for identifying polarized osteoclasts

Osteoclasts form ruffled borders and sealing zones toward bone surfaces to resorb bone. Sealing zones are defined as ringed structures of F-actin dots (actin rings). Polarized osteoclasts secrete protons to bone surfaces via vacuolar proton ATPase through ruffled borders. Catabolic enzymes such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K are also secreted to bone surfaces. Here we show a simple method of identifying functional vestiges of polarized osteoclasts. Osteoclasts obtained from cocultures of mouse osteoblasts and bone marrow cells were cultured for 48 h on dentin slices. Cultures were then fixed and stained for TRAP to identify osteoclasts on the slices. Cells were removed from the slices with cotton swabs, and the slices subjected to TRAP and Mayer's hematoxylin staining. Small TRAP-positive spots (TRAP-marks) were detected in the resorption pits stained with Mayer's hematoxylin. Pitted areas were not always located in the places of osteoclasts, but osteoclasts existed on all TRAP-marks. A time course experiment showed that the number of TRAP-marks was maintained, while the number of resorption pits increased with the culture period. The position of actin rings formed in osteoclasts corresponded to that of TRAP-marks on dentin slices. Immunostaining of dentin slices showed that both cathepsin K and vacuolar proton ATPase were colocalized with the TRAP-marks. Treatment of osteoclast cultures with alendronate, a bisphosphonate, suppressed the formation of TRAP-marks and resorption pits without affecting the cell viability. Calcitonin induced the disappearance of both actin rings and TRAP-marks in osteoclast cultures. These results suggest that TRAP-marks are vestiges of proteins secreted by polarized osteoclasts.     

Immunolocalization of matrix metalloproteinase-13 on bone surface under osteoclasts in rat tibia

Matrix metalloproteinase (MMP)-13 (an interstitial collagenase also called collagenase 3) is involved in degradation of extracellular matrix in various tissues. Using immunohistochemistry and Western blotting, we investigated localization of MMP-13 in rat tibia, to clarify the role of MMP-13 in bone resorption. MMP-13 reactivity was mainly seen on bone surfaces under osteoclasts, and in some osteocytes and their lacunae near osteoclasts. However, immunoreactivity was not seen in chondrocytes or osteoclasts. MMP-13 was also localized on cement lines in the epiphysis. In the growth plate erosion zone, perivascular cells showed MMP-13 reactivity. Immunoelectron microscopy revealed that MMP-13 was localized on the bone surfaces, under the ruffled borders and some clear zones of osteoclasts. Gold-labeled MMP-13 was closely associated with collagen fibrils. Gold labeling was also detected in Golgi apparatus of osteocytes adjacent to osteoclasts and bone lining cells. Western blotting showed that MMP-13 was mainly associated with mineralized bone matrix. These findings suggest that MMP-13 synthesized and secreted by osteoblast-lineage cells is localized under the ruffled borders of osteoclasts. MMP-13 may play an important role in degradation of type I collagen in bone matrix, acting in concert with cathepsin K and MMP-9 produced by osteoclasts. MMP-13 in perivascular cells may be involved in removal of cartilage matrix proteins such as type II collagen and aggrecan.     

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.     

A peptidyl derivative structurally based on the inhibitory center of cystatin C inhibits bone resorption in vitro

Human cystatin C is a cysteine proteinase inhibitor belonging to the cystatin superfamily, which previously has been shown to inhibit bone resorption in bone organ culture. The aminoterminal segment, Arg(8)-Leu(9)-Val(10)-Gly(11) (RLVG), of the single polypeptide chain of cystatin C constitutes an essential part of its inhibitory center. In the present study, the effect of benzyloxycarbonyl-Arg(8)-Leu(9)-Val(10)-Gly(11)-diazomethane (Z-RLVG-CHN(2)) on bone resorption in vitro was compared with the effects of cystatin C and calcitonin. Bone resorption was assessed by the release of (45)Ca and (3)H from mouse calvarial bones prelabeled with [(45)Ca]CaCl(2) and [(3)H]-proline, respectively. Z-RLVG-CHN(2) concentration-dependently inhibited the release of (45)Ca and (3)H in bones stimulated by parathyroid hormone (PTH), with half-maximal inhibition obtained at 1 micromol/L. The inhibitory actions of Z-RLVG-CHN(2) and cystatin C were persistent, whereas action induced initially by calcitonin was lost with time. The inhibition caused by Z-RLVG-CHN(2) and cystatin C on PTH-stimulated (45)Ca release was observed after 6 h, whereas inhibition by calcitonin was seen already after 2 h. In contrast, the inhibitory effects of Z-RLVG-CHN(2) and cystatin C, as well as that of calcitonin, on (3)H release was seen already after 2 h. Z-RLVG-CHN(2), in which the reactive carboxyterminal diazomethane was substituted by nonreactive groups [-OH, -NH(2), or -N(CH(3))(2)], resulted in peptidyl derivatives, which, in contrast to Z-RLVG-CHN(2) and cystatin C, inhibited neither cysteine proteinases nor bone resorption. In contrast to wild-type cystatin C, recombinant human cystatin C with Gly substitutions for residues Arg(8), Leu(9), Val(10), and Trp(106), and with low or nonexistent affinity for cysteine proteinases, did not display any inhibitory effect on bone resorption. These data strongly indicate that Z-RLVG-CHN(2) inhibits bone resorption in vitro by a mechanism that seems primarily to be due to an inhibition of bone matrix degradation via cysteine proteinases. The data also corroborate the hypothesis that cystatin C inhibits bone resorption by virtue of its cysteine proteinase inhibitory capacity.     

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.