A scrutiny of matrix metalloproteinases in osteoclasts: evidence for heterogeneity and for the presence of MMPs synthesized by other cells

Genetic diseases and knockout mice stress the importance of matrix metalloproteinases (MMPs) in skeletal turnover. Our study aims at clarifying which MMPs are expressed by osteoclasts. Previous analyses of this basic question led to conflicting reports in the literature. In the present study, we used a variety of approaches: PCR, Northern blots, Slot blots, in situ hybridization, and immunohistochemistry. We analyzed osteoclasts in culture as well as osteoclasts in native bone at different locations and compared mouse and rabbit osteoclasts. Osteoclasts express MMP-9 and -14 in all conditions, although to a variable extent, and they are able to synthesize MMP-3, -10, and -12, at least under some circumstances. The induction of a given MMP in osteoclasts is influenced by its environment (e.g., osteoclast culture vs. native bone, and various sites within the same bone) and depends on the species (e.g., mouse vs. rabbit). Osteoclasts show high amounts of MMP-2 and -13 protein presumably made to a large extent by other cells, thereby documenting how proteinases of nonosteoclastic origin may contribute to osteoclast activities and giving insight in why the resorptive activity of purified osteoclasts appears insensitive to MMP inhibitors. Our study shows that the confusion about osteoclastic MMPs in the literature reflects the remarkable ability of osteoclasts to adapt to their environment, as required by the structural or functional diversity of bone tissue. Our observations provide basic information needed for understanding the emerging role of MMPs in controlling cell signaling and bone resorption.     

A selective inhibitor of matrix metalloproteinases inhibits the migration of isolated osteoclasts by increasing the life span of podosomes

The osteoclast is a unique cell that cycles between bone resorption and migration. In this study, we used KB-R7785, an inhibitor of matrix metalloproteinases (MMPs), to investigate the role of MMPs in this cycle. Osteoclasts prepared from neonatal rabbits were processed to measure: (1) migration on hydroxyapatite (HA)-coated dishes, (2) the formation of an actin band, which is a large collection of podosomes, (3) the life span of podosomes, and (4) lacunar resorption on ivory slices. KB-R7785 significantly decreased the area of the tracks formed by osteoclasts on HA-coated dishes. The percentage of actin/podosome band formation, expressed as: (total length of the actin band/circumference of the osteoclast) × 100, was significantly increased after the addition of KB-R7785, which implies that podosome disassembly in osteoclasts was decreased by the inhibition of MMPs. Time-lapse cinemicrography demonstrated that the addition of KB-R7785 increased the life span of podosomes. Several indexes (number of resorption pits, total resorption area, and mean resorption area) of osteoclast resorption activity were significantly decreased by KB-R7785, while the average depth of resorption was increased. These results indicate that decreased podosome disassembly caused by an MMP inhibitor suppresses the migration of osteoclasts and, ultimately, inhibits osteoclastic bone resorption. Received: April 11, 2001 / Accepted: October 22, 2001     

Transforming growth factor-beta induces osteoclast ruffling and chemotaxis: potential role in osteoclast recruitment.

Transforming growth factor-beta (TGF-beta) is released from the matrix during bone resorption and has been implicated in the pathogenesis of giant cell tumors of bone and the expansion of breast cancer metastases in bone. Because osteoclasts mediate tumor-induced osteolysis, we investigated whether TGF-beta stimulates osteoclast recruitment. Osteoclasts were isolated from rat long bones and time-lapse video microscopy was used to monitor their morphology and motility. Within 5 minutes, TGF-beta (0.1 nM) induced dynamic ruffling, with 65% of osteoclasts displaying membrane ruffles compared with 35% in untreated controls. Over a 2-h period, osteoclasts exhibited significant directed migration toward a source of TGF-beta, indicating chemotaxis. echistatin, an alphavbeta3 integrin blocker that inhibits macrophage colony-stimulating factor (M-CSF)-induced osteoclast migration, did not prevent the migration of osteoclasts toward TGF-beta. In contrast, a beta1 integrin blocking antibody inhibited osteoclast chemotaxis toward TGF-beta but not M-CSF. These data indicate the selective use of integrins by osteoclasts migrating in response to different chemotaxins. In addition, wortmannin and U0126 inhibited TGF-beta-induced chemotaxis, suggesting involvement of the phosphatidylinositol 3 (PI 3) kinase and mitogen-activated protein (MAP) kinase signaling pathways. Physiologically, TGF-beta, may coordinate osteoclast activity by recruiting osteoclasts to existing sites of resorption. Pathologically, TGF-beta-induced osteoclast recruitment may be critical for expansion of primary and metastatic tumors in bone.     

Chemically modified tetracyclines act through multiple mechanisms directly on osteoclast precursors

Chemically modified tetracyclines (CMTs) are thought to inhibit bone resorption primarily through their ability to inhibit matrix metalloproteinases (MMPs). We have previously demonstrated that some tetracycline compounds (TCs) induce apoptosis in mature rabbit osteoclasts and inhibit osteoclastic resorption in mouse osteoblast/marrow co-cultures in vitro. In this report, we now show that non-antibiotic analogues of doxycycline (CMT-3) and minocycline (CMT-8) are potent inhibitors of osteoclastogenesis in vitro from human peripheral blood mononuclear cells (PBMC) stimulated with macrophage colony stimulating factor (MCSF) and receptor activator of NF-kappaB ligand (RANKL), through an action that is independent of osteoblast-osteoclast interactions. Osteoclast formation over 20 days was completely abrogated when CMT-3 or CMT-8 were included in PBMC cultures at a concentration of 250 ng/ml, although doxycycline at this concentration reduced osteoclast formation to ca. 50% of control. CMT-3 and CMT-8 also significantly induced apoptosis over 24 h in mature osteoclasts generated over 20 days when added to cultures at 5 microg/ml or more. In a time-course experiment, apoptosis was evident after a delay of 1-2 h following treatment of mature osteoclasts with CMT-3 at 20 microg/ml. The broad-spectrum MMP inhibitor BB94 (Batimastat) did not recapitulate the apoptosis induced by CMT-3, even at a concentration where MMP-13 activity was completely inhibited. There was no evidence for an anabolic effect of any of the TCs on osteoblast lineage cells in a calcifying fibroblastic colony (CFU-f) formation assay, where CMT-3 partially inhibited CFU-f formation at 5 microg/ml. Our data indicate that inhibition of osteoclast formation and induction of osteoclast apoptosis are pharmacologically significant actions of CMTs in inhibiting bone resorption, and that osteoclast apoptosis cannot be attributed to the ability of CMTs to inhibit MMPs or to actions mediated by osteoblastic lineage cells.     

Osteoclast TGF‐β Receptor Signaling Induces Wnt1 Secretion and Couples Bone Resorption to Bone Formation

Osteoblast‐mediated bone formation is coupled to osteoclast‐mediated bone resorption. These processes become uncoupled with age, leading to increased risk for debilitating fractures. Therefore, understanding how osteoblasts are recruited to sites of resorption is vital to treating age‐related bone loss. Osteoclasts release and activate TGF‐β from the bone matrix. Here we show that osteoclast‐specific inhibition of TGF‐β receptor signaling in mice results in osteopenia due to reduced osteoblast numbers with no significant impact on osteoclast numbers or activity. TGF‐β induced osteoclast expression of Wnt1, a protein crucial to normal bone formation, and this response was blocked by impaired TGF‐β receptor signaling. Osteoclasts in aged murine bones had lower TGF‐β signaling and Wnt1 expression in vivo. Ex vivo stimulation of osteoclasts derived from young or old mouse bone marrow macrophages showed no difference in TGF‐β–induced Wnt1 expression. However, young osteoclasts expressed reduced Wnt1 when cultured on aged mouse bone chips compared to young mouse bone chips, consistent with decreased skeletal TGF‐β availability with age. Therefore, osteoclast responses to TGF‐β are essential for coupling bone resorption to bone formation, and modulating this pathway may provide opportunities to treat age‐related bone loss. © 2015 American Society for Bone and Mineral Research.     

Transmigration: a new property of mature multinucleated osteoclasts.

Even though it is assumed that multinucleated osteoclasts are migrating cells on the bone surface to be resorbed, we show that they can also selectively transmigrate through layers of cells usually found in the bone microenvironment. This activity is associated with c-src and MMPs and can be stimulated by bone metastatic breast cancer cells, a process blocked by bisphosphonate treatment. INTRODUCTION: Osteoclasts have an hematopoietic origin and are bone-resorbing cells. Monocytic precursors migrate to the bone surface where they fuse to form multinucleated osteoclasts able to migrate over the bone surface. We studied whether multinucleated osteoclasts were also able to transmigrate through tissues. MATERIALS AND METHODS: Murine spleen-derived and green fluorescent protein (GFP)-Raw derived osteoclasts were seeded on osteoblasts and several other cell types. The cells were fixed for 20 minutes, 4 or 12 h after osteoclast seeding, and stained with phalloidin to visualize actin using confocal microscopy. Drugs such as PP2 and GM6001, inhibitors of c-src and matrix metalloproteinases (MMPs), respectively, and risedronate were used to determine osteoclast transmigration regulating factors. RESULTS: We observed by confocal microscopy that multinucleated osteoclasts specifically transmigrate through confluent layers of various cell types present in the bone microenvironment in vitro. This is an efficient process associated with c-src and MMPs but is independent of podosomes. Moreover, conditioned medium from bone metastatic breast cancer cells stimulates osteoclast transmigration in vitro, a process inhibited by bisphosphonate treatment. CONCLUSIONS: Our data describe a new property of mature multinucleated osteoclasts to transmigrate through various cell types. The ability to control this highly regulated osteoclast transmigration process may offer new therapeutic strategies for bone diseases associated with an imbalance in bone remodeling caused by excessive osteoclast resorption.     

Protein geranylgeranylation is required for osteoclast formation, function, and survival: inhibition by bisphosphonates and GGTI-298.

Bisphosphonates are the important class of antiresorptive drugs used in the treatment of metabolic bone diseases. Although their molecular mechanism of action has not been fully elucidated, recent studies have shown that the nitrogen-containing bisphosphonates can inhibit protein prenylation in macrophages in vitro. In this study, we show that the nitrogen-containing bisphosphonates risedronate, zoledronate, ibandronate, alendronate, and pamidronate (but not the non nitrogen-containing bisphosphonates clodronate, etidronate, and tiludronate) prevent the incorporation of [14C]mevalonate into prenylated (farnesylated and geranylgeranylated) proteins in purified rabbit osteoclasts. The inhibitory effect of nitrogen-containing bisphosphonates on bone resorption is likely to result largely from the loss of geranylgeranylated proteins rather than loss of farnesylated proteins in osteoclasts, because concentrations of GGTI-298 (a specific inhibitor of geranylgeranyl transferase I) that inhibited protein geranylgeranylation in purified rabbit osteoclasts prevented osteoclast formation in murine bone marrow cultures, disrupted the osteoclast cytoskeleton, inhibited bone resorption, and induced apoptosis in isolated chick and rabbit osteoclasts in vitro. By contrast, concentrations of FTI-277 (a specific inhibitor of farnesyl transferase) that prevented protein farnesylation in purified rabbit osteoclasts had little effect on osteoclast morphology or apoptosis and did not inhibit bone resorption. These results therefore show the molecular mechanism of action of nitrogen-containing bisphosphonate drugs in osteoclasts and highlight the fundamental importance of geranylgeranylated proteins in osteoclast formation and function.     

Human mesenchymal tumour-associated macrophages differentiate into osteoclastic bone-resorbing cells

The cellular mechanisms which account for the formation of osteoclasts and bone resorption associated with enlarging benign and malignant mesenchymal tumours of bone are uncertain. Osteoclasts are marrow-derived, multinucleated, bone-resorbing cells which express a macrophage phenotype. We have determined whether tumour-associated macrophages (TAMs) isolated from benign and malignant mesenchymal tumours are capable of differentiating into osteoclasts. Macrophages were cultured on both coverslips and dentine slices for up to 21 days with UMR 106 osteoblastic cells in the presence of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) and human macrophage colony-stimulating factor (M-CSF) or, in the absence of UMR 106 cells, with M-CSF and RANK ligand. In all tumours, the formation of osteoclasts from CD14-positive macrophages was shown by the formation of tartrate-resistant-acid-phosphatase and vitronectin-receptor-positive multinucleated cells which were capable of carrying out lacunar resorption. These results indicate that the tumour osteolysis associated with the growth of mesenchymal tumours in bone is likely to be due in part to the differentiation of mononuclear phagocyte osteoclast precursors which are present in the TAM population of these lesions.     

Inhibition of the classical NF-κB pathway prevents osteoclast bone-resorbing activity

The classical NF-κB pathway plays an important role in osteoclast formation and differentiation; however, the role of NF-κB in osteoclast bone-resorbing activity is not well understood. To elucidate whether NF-κB is important for osteoclast bone-resorbing activity, we used a selective peptide inhibitor of the classical NF-κB pathway named the NBD peptide. Osteoclasts were generated using bone marrow macrophages in the presence of M-CSF and RANKL. The NBD peptide dose-dependently blocked the bone-resorbing activity of osteoclasts by reducing area, volume (p < 0.001) and depths (p < 0.05) of pits. The reduced resorption by the peptide was due to reduced osteoclast bone-resorbing activity, but not reduced differentiation as the number of osteoclasts was similar in all groups. The peptide inhibited bone resorption by reducing TRAP activity, disrupting actin rings and preventing osteoclast migration. Gene expressions of a panel of bone resorption markers were significantly reduced. The NBD peptide dose-dependently reduced the RANKL-induced c-Src kinase activity, which is important for actin ring formation and osteoclast bone resorption. Therefore, these data suggest that the classical NF-κB pathway plays a pivotal role in osteoclast bone-resorbing activity.     

A specific subtype of osteoclasts secretes factors inducing nodule formation by osteoblasts

Osteoclasts are known to be important for the coupling process between bone resorption and formation. The aim of this study was to address when osteoclasts are anabolically active. Human monocytes were differentiated into mature osteoclasts by treatment with M-CSF and RANKL. Conditioned medium was collected from macrophages, pre-osteoclasts, and mature functional or non-resorbing osteopetrotic osteoclasts on either bone, plastic, decalcified bone or dentine with or without diphyllin, E64 or GM6001. Osteoclasts numbers were measured by TRACP activity. Bone resorption was evaluated by CTX-I and calcium release. The osteoblastic cell line 2T3 was treated with 50% of CM or non-CM for 12days. Bone formation was assessed by Alizarin Red extraction. CM from mature osteoclasts induced bone formation, while CM from macrophages did not. Non-resorbing osteoclasts generated from osteopetrosis patients showed little resorption, but still an induction of bone formation by osteoblasts. Mimicking the reduction in bone resorption using the V-ATPase inhibitor Diphyllin, the cysteine proteinase inhibitor E64 and the MMP-inhibitor GM6001 showed that CM from diphyllin and E64 treated osteoclasts showed reduced ability to induce bone formation compared to CM from vehicle treated osteoclasts, while CM from GM6001 treated osteoclasts equaled vehicle CM. Osteoclasts on either dentine or decalcified bone showed strongly attenuated anabolic capacities. In conclusion, we present evidence that osteoclasts, both dependent and independent of their resorptive activity, secrete factors stimulating osteoblastic bone formation.     

Reactive oxygen species and oxidative stress in osteoclastogenesis,skeletal aging and bone diseases

Osteoclasts are cells derived from bone marrow macrophages and are important in regulating bone resorption during bone homeostasis. Understanding what drives osteoclast differentiation and activity is important when studying diseases characterized by heightened bone resorption relative to formation, such as osteoporosis. In the last decade, studies have indicated that reactive oxygen species (ROS), including superoxide and hydrogen peroxide, are crucial components that regulate the differentiation process of osteoclasts. However, there are still many unanswered questions that remain. This review will examine the mechanisms by which ROS can be produced in osteoclasts as well as how it may affect osteoclast differentiation and activity through its actions on osteoclastogenesis signaling pathways. In addition, the contribution of ROS to the aging-associated disease of osteoporosis will be addressed and how targeting ROS may lead to the development of novel therapeutic treatment options.     

Inhibition of osteoclastogenesis by RNA interference targeting RANK

ABSTRACT: BACKGROUND: Osteoclasts and osteoblasts regulate bone resorption and formation to allow bone remodeling and homeostasis. The balance between bone resorption and formation is disturbed by abnormal recruitment of osteoclasts. Osteoclast differentiation is dependent on the receptor activator of nuclear factor NF-kappa B (RANK) ligand (RANKL) as well as the macrophage colony-stimulating factor (M-CSF). The RANKL/RANK system and RANK signaling induce osteoclast formation mediated by various cytokines. The RANK/RANKL pathway has been primarily implicated in metabolic, degenerative and neoplastic bone disorders or osteolysis. The central role of RANK/RANKL interaction in osteoclastogenesis makes RANK an attractive target for potential therapies in treatment of osteolysis. The purpose of this study was to assess the effect of inhibition of RANK expression in mouse bone marrow macrophages on osteoclast differentiation and bone resorption. METHODS: Three pairs of short hairpin RNAs (shRNA) targeting RANK were designed and synthesized. The optimal shRNA was selected among three pairs of shRNAs by RANK expression analyzed by Western blot and Real-time PCR. We investigated suppression of osteoclastogenesis of mouse bone marrow macrophages (BMMs) using the optimal shRNA by targeting RANK. RESULTS: Among the three shRANKs examined, shRANK-3 significantly suppressed [88.3%] the RANK expression (p < 0.01). shRANK-3 also brought about a marked inhibition of osteoclast formation and bone resorption as demonstrated by tartrate--resistant acid phosphatase (TRAP) staining and osteoclast resorption assay. The results of our study show that retrovirus-mediated shRANK-3 suppresses osteoclast differentiation and osteolysis of BMMs. CONCLUSIONS: These findings suggest that retrovirus-mediated shRNA targeting RANK inhibits osteoclast differentiation and osteolysis. It may appear an attractive target for preventing osteolysis in humans with a potential clinical application.     

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.     

Role of DC-STAMP in cellular fusion of osteoclasts and macrophage giant cells

Osteoclasts are the only cells that can resorb bone matrix physiologically and maintain the bone content. Osteoclasts are derived from macrophage/monocyte lineage cells; stimulation by macrophage colony stimulating factor and receptor activator of NFκB ligand induces osteoclastogenesis. During osteoclastogenesis, preosteoclasts fuse to form multinuclear mature osteoclasts. Cellular fusion is a unique phenomenon and enables fertilization, myotube formation, and efficient bone resorption in vertebrates. To date, several molecules have been reported to be fusion related in osteoclasts, namely CD44, CD47, ADAM12, MCP-1, and CD9, although the molecules which regulate osteoclast cellular fusion remain unclear. Here, we show that the seven-transmembrane-region receptor dendritic cell-specific transmembrane protein (DC-STAMP) is required for cell–cell fusion of osteoclasts and foreign body giant cells.     

Alendronate Disturbs Vesicular Trafficking in Osteoclasts

The nitrogen-containing bisphosphonate alendronate inhibits osteoclast-mediated bone resorption through inhibition of the mevalonate pathway. This results in impaired protein prenylation and may affect the function of small GTPases in osteoclasts. Since these proteins are important regulators of vesicle transport in cells, we investigated the possible interference of alendronate with these processes in isolated rat osteoclasts. We show here that alendronate-induced inhibition of bone resorption coincides with accumulation of tartrate-resistant acid phosphatase- and electron dense material-containing tubular vesicles in osteoclasts. Alendronate-induced changes in osteoclasts also included widening of the sealing zone areas and incomplete organization of tight attachments and ruffled borders. Osteoclasts also appeared partially detached from the bone surface, and organic matrix was typically dissolved only at the edges of the resorption pits on alendronate-coated bone slices. In contrast, resorption pits on the control and clodronate-coated bone slices were thoroughly resorbed. Inhibition of bone resorption by alendronate was not, however, related to a decrease in osteoclast number. In conclusion, our findings suggest that alendronate inactivates osteoclasts by mechanisms that impair their intracellular vesicle transport, apoptosis being only a secondary phenomenon to this.     

Bovine dentine organic matrix down-regulates osteoclast activity

Physiological root resorption is a phenomenon that normally takes place in deciduous teeth; root resorption of permanent teeth occurs only under pathological conditions. The molecular mechanisms underlying these processes are still unclear. Our previous study showed that osteoclasts cultured on deciduous dentine exhibited a higher degree of resorption and higher levels of cathepsin K and MMP-9 mRNA than osteoclasts cultured on permanent dentine. These results could be because of different susceptibilities to acid and the different organic matrices between deciduous and permanent dentine. Thus, the purpose of this study was to investigate the effect of dentine extracts from bovine deciduous and permanent dentine on osteoclast activity. Osteoclasts, obtained from mouse bone marrow cells co-cultured with an osteoblast-rich fraction in the presence of 1,25-(OH)₂-vitamin D3 and PGE2, were incubated with or without 0.6 M HCl extracts from bovine deciduous or permanent dentine for 48 h. TRAP positive cell number, TRAP activity, the areas of resorption pits, and mRNA levels of TRAP, v-ATPase, calcitonin receptor, cathepsin K, and MMP-9 were examined. The results illustrated that TRAP activity, the resorbed area, and the mRNA levels of osteoclast marker genes seemed to be suppressed by both deciduous and permanent dentine extracts. These findings indicate that some factors that suppress osteoclast activity are contained in both deciduous and permanent dentine extracts. Although there was no significant difference in osteoclast activity between deciduous and permanent dentine extracts, osteoclasts incubated with permanent dentine extracts tend to exhibit less resorption activity than those incubated with deciduous dentine extracts. However, we could not clearly explain the causes of this.     

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.     

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.