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.     

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.     

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.     

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

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

The R740S mutation in the V‐ATPase a3 subunit increases lysosomal pH,impairs NFATc1 translocation,and decreases in vitro osteoclastogenesis

Vacuolar H⁺‐ATPase (V‐ATPase), a multisubunit enzyme located at the ruffled border and in lysosomes of osteoclasts, is necessary for bone resorption. We previously showed that heterozygous mice with an R740S mutation in the a3 subunit of V‐ATPase (+/R740S) have mild osteopetrosis resulting from an ～90% reduction in proton translocation across osteoclast membranes. Here we show that lysosomal pH is also higher in +/R740S compared with wild‐type (+/+) osteoclasts. Both osteoclast number and size were decreased in cultures of +/R740S compared with +/+ bone marrow cells, with concomitant decreased expression of key osteoclast markers (TRAP, cathepsin K, OSCAR, DC‐STAMP, and NFATc1), suggesting that low lysosomal pH plays an important role in osteoclastogenesis. To elucidate the molecular mechanism of this inhibition, NFATc1 activation was assessed. NFATc1 nuclear translocation was significantly reduced in +/R740S compared with +/+ cells; however, this was not because of impaired enzymatic activity of calcineurin, the phosphatase responsible for NFATc1 dephosphorylation. Protein and RNA expression levels of regulator of calcineurin 1 (RCAN1), an endogenous inhibitor of NFATc1 activation and a protein degraded in lysosomes, were not significantly different between +/R740S and +/+ osteoclasts, but the RCAN1/NFATc1 ratio was significantly higher in +/R740S versus +/+ cells. The lysosomal inhibitor chloroquine significantly increased RCAN1 accumulation in +/+ cells, consistent with the hypothesis that higher lysosomal pH impairs RCAN1 degradation, leading to a higher RCAN1/NFATc1 ratio and consequently NFATc1 inhibition. Our data indicate that increased lysosomal pH in osteoclasts leads to decreased NFATc1 signaling and nuclear translocation, resulting in a cell autonomous impairment of osteoclastogenesis in vitro. © 2013 American Society for Bone and Mineral Research     

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.     

Critical role of cortactin in actin ring formation and osteoclastic bone resorption

Tyrosine kinase c-Src plays an essential role in ruffled border formation and bone resorption in osteoclasts; however, it is unclear how c-Src controls ruffled border formation during bone resorption. To address this question, we investigated the role of cortactin, a c-Src substrate, in osteoclasts. We found that cortactin showed colocalization with c-Src and actin rings in osteoclasts. Overexpression of cortactin stimulated actin ring formation in RAW 264.7 cells. In contrast, overexpression of Csk inhibited tyrosine phosphorylation of cortactin and binding of cortactin to c-Src. More importantly, overexertion of a mutant cortactin strongly suppressed actin ring formation and bone resorbing activity in osteoclasts. Collectively, our data indicate that cortactin controls osteoclastic bone resorption by regulating actin organization.     

Disruption of the dynein‐dynactin complex unveils motor‐specific functions in osteoclast formation and bone resorption

Osteoclastic bone resorption requires strict interplay between acidified carrier vesicles, motor proteins, and the underlying cytoskeleton in order to sustain the specialized structural and functional polarization of the ruffled border. Cytoplasmic dynein, a large processive mechanochemical motor comprising heavy, intermediate, and light chains coupled to the dynactin cofactor complex, powers unilateral motility of diverse cargos to microtubule minus‐ends. We have recently shown that regulators of the dynein motor complex constitute critical components of the osteoclastic bone resorptive machinery. Here, by selectively modulating endogenous dynein activity, we show that the integrity of the dynein‐dynactin motor complex is an essential requirement for both osteoclast formation and function. Systematic dissection of the osteoclast dynein‐dynactin complex revealed that it is differentially localized throughout RANKL‐induced osteoclast formation and activation, undergoing microtubule‐coupled reorganization upon the establishment of cellular polarization. In osteoclasts actively resorbing bone, dynein‐dynactin intimately co‐localizes with the CAP‐Gly domain‐containing microtubule plus‐end protein CLIP‐170 at the resorptive front, thus orientating the ruffled border as a microtubule plus‐end domain. Unexpectedly, disruption of the dynein‐dynactin complex by exogenous p50/dynamitin expression retards osteoclast formation in vitro, owing largely to prolonged mitotic stasis of osteoclast progenitor cells. More importantly, loss of osteoclastic dynein activity results in a drastic redistribution of key intracellular organelles, including the Golgi and lysosomes, an effect that coincides with impaired cathepsin K secretion and diminished bone resorptive function. Collectively, these data unveil a previously unrecognized role for the dynein‐dynactin motor complex in osteoclast formation and function, serving not only to regulate their timely maturation but also the delivery of osteolytic cargo that is essential to the bone resorptive process. © 2013 American Society for Bone and Mineral Research     

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.     

Polarization and secretion of cathepsin K precede tartrate-resistant acid phosphatase secretion to the ruffled border area during the activation of matrix-resorbing clasts

The activation sequence of clasts (the designation clast was used because ultrastructurally in this tissue, it is not always possible to differentiate between chondroclasts sitting on cartilage and osteoclasts sitting on bone matrix) was studied in vivo using the healing of low-phosphate, vitamin D-deficiency rickets as a model system. Thus, the bones of 7-week-old rachitic animals were analyzed with a combination of morphological, biochemical, and molecular biological methods at 48 and 72 h, respectively, after change to normal food. A quantitative ultrastructural analysis showed that the number of clast profiles exhibiting the characteristic polarized features of actively resorbing cells, i.e., ruffled borders and clear zones, had reached normal levels after 48 h. By combining the data with quantitative analyses by the immunogold technique, we demonstrated that cathepsin K secretion was coupled to ruffled border formation in clasts irrespective of whether the number of polarized clasts was low (in rickets) or high (in healing). In contrast, the levels of tartrate-resistant acid phosphatase (TRAP) both between ruffles and in the outside matrix adjoining the ruffled border were low in polarized clasts both in rickets and at the early (48 h) healing time-point, but were increased at the latest (72 h) healing time-point. Interestingly, expression of TRAP and the cathepsin K at the mRNA level, as well as protein expression and the activity of TRAP, were not different during the healing sequence. Although the two enzymes are confined to the same clast populations, their secretion during the resorption process is apparently differentially regulated: cathepsin K secretion is coupled to ruffled border formation in clasts, whereas TRAP is secreted at a later stage during the resorption sequence, suggesting a role for secreted TRAP as a modulator of resorptive activity.     

Class IA phosphatidylinositol 3‐kinase regulates osteoclastic bone resorption through protein kinase B–mediated vesicle transport

Class IA phosphatidylinositol 3‐kinases (PI3Ks) are activated by growth factor receptors and regulate a wide range of cellular processes. In osteoclasts, they are activated downstream of α_vβ₃ integrin and colony‐stimulating factor‐1 receptor (c‐Fms), which are involved in the regulation of bone‐resorbing activity. The physiological relevance of the in vitro studies using PI3K inhibitors has been of limited value, because they inhibit all classes of PI3K. Here, we show that the osteoclast‐specific deletion of the p85 genes encoding the regulatory subunit of the class IA PI3K results in an osteopetrotic phenotype caused by a defect in the bone‐resorbing activity of osteoclasts. Class IA PI3K is required for the ruffled border formation and vesicular transport, but not for the formation of the sealing zone. p85α/β doubly deficient osteoclasts had a defect in macrophage colony‐stimulating factor (M‐CSF)–induced protein kinase B (Akt) activation and the introduction of constitutively active Akt recovered the bone‐resorbing activity. Thus, the class IA PI3K‐Akt pathway regulates the cellular machinery crucial for osteoclastic bone resorption, and may provide a molecular basis for therapeutic strategies against bone diseases. © 2013 American Society for Bone and Mineral Research.     

Ultrastructural localization of a tartrate-resistant acid ATPase in bone

Osteoclasts are effector cells in bone breakdown, and the active bone resorption is confined to the ruffled border zone of these cells. An acid milieu is maintained in this zone which is probably a prerequisite for bone resorption. Tartrate-resistant acid phosphatase (TRAP) activity has been recognized as a characteristic property of osteoclasts and in several studies proposed as a cytochemical marker of osteoclasts. We have previously isolated and characterized a tartrate-resistant and iron-activated acid ATPase (TrATPase) from rat bone, the enzyme being a member of the TRAP family. In the present study the ultrastructural localization of this enzyme was delineated by employing immunogold technique on low temperature-embedded maxillar rat bone. Intensive immunolabeling was seen on the bone surfaces facing the ruffled border zone while lower amounts of marker were seen in adjacent bone areas, that is, on the bone surfaces facing the clear zone and deeper-into the bone. Within the osteoclasts gold markers were observed mainly in vesicular structures interpreted as lysosomes. Immunolabeling was also observed in the recently described endocytic cells located near osteoblasts and osteoclasts. Also in these cells, the marker was confined to lysosomelike structures. The amount of label in bone facing osteoblasts was low, as was the amount within osteoblasts. Our observation of extracellular localization, in particular accumulation of TrATPase in bone matrix facing the ruffled border area of the osteoclasts, favors the view that the enzyme is exported to areas of active bone resorption, thereby indicating a potential role for the enzyme in this process.     

The V–ATPase a3 subunit mutation R740S is dominant negative and results in osteopetrosis in mice

A mouse founder with high bone mineral density and an osteopetrotic phenotype was identified in an N‐ethyl‐N‐nitrosourea (ENU) screen. It was found to carry a dominant missense mutation in the Tcirg1 gene that encodes the a3 subunit of the vacuolar type H⁺‐ATPase (V–ATPase), resulting in replacement of a highly conserved amino acid (R740S). The +/R740S mice have normal appearance, size, and weight but exhibit high bone density. Osteoblast parameters are unaffected in bones of +/R740S mice, whereas osteoclast number and marker expression are increased, concomitant with a decrease in the number of apoptotic osteoclasts. Consistent with reduced osteoclast apoptosis, expression of Rankl and Bcl2 is elevated, whereas Casp3 is reduced. Transmission electron microscopy revealed that unlike other known mutations in the a3 subunit of V–ATPase, polarization and ruffled border formation appear normal in +/R740S osteoclasts. However, V–ATPases from +/R740S osteoclast membranes have severely reduced proton transport, whereas ATP hydrolysis is not significantly affected. We show for the first time that a point mutation within the a3 subunit, R740S, which is dominant negative for proton pumping and bone resorption, also uncouples proton pumping from ATP hydrolysis but has no effect on ruffled border formation or polarization of osteoclasts. These results suggest that the V₀ complex has proton‐pumping‐independent functions in mammalian cells. © 2011 American Society for Bone and Mineral Research.     

Expression and regulation of RAB3 proteins in osteoclasts and their precursors.

The ruffled membrane, the resorptive organelle of the osteoclast, is generated by fusion of intracytoplasmic acidifying vesicles with the plasma membrane, an event analogous to regulated exocytosis. While the ruffled membrane is essential to the bone resorptive process, the mechanisms governing its generation are unknown. However, regulated exocytosis is mediated, in part, by isoforms of the Rab3 subset of Rab GTPases. Because of similarities between exocytosis and ruffled membrane formation, we asked if Rab3 proteins are expressed by osteoclasts or their precursors, and if so, are these molecules regulated by agents known to prompt the osteoclast phenotype? We find murine osteoclast precursors, in the form of bone marrow macrophages (BMMs), express at least two Rab3 isoforms, namely A and B/C, which are individually enhanced by a variety of hematopoietic cytokines. Consistent with the osteoclastogenic properties of a number of these cytokines, differentiation of BMMs into osteoclasts, in vitro, is associated with increased expression of both isoforms, particularly Rab3B/C. Finally, Rab3B/C localizes with the avian osteoclast H+ATPase (vacuolar proton pump) and pp60c-src, both intracellularly and within acidifying vesicles derived largely from the ruffled membrane. Thus, expression of specific rab3 proteins, an event which may control formation of the osteoclast ruffled membrane, is modulated by cytokines during osteoclastogenesis.     

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.     

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.     

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.     

Cbp recruitment of Csk into lipid rafts is critical to c‐Src kinase activity and bone resorption in osteoclasts

A tyrosine kinase, c‐Src, that plays an indispensable role in ruffled border formation and bone resorption is constitutively active in osteoclasts. However, to date, the molecular mechanism underlying increased c‐Src activity in osteoclasts is unknown. To address this, we first examined the expression levels and subcellular localization of Csk, a negative regulatory kinase for c‐Src. We found that the expression level of Csk in osteoclasts was comparable with that of other tissues. However, in osteoclasts, Csk was hardly localized in lipid rafts, where c‐Src is highly expressed. Interestingly, expression of Cbp, which recruits Csk into lipid rafts through physical interaction with Csk, was very low in osteoclasts compared with other tissues. To understand the importance of Cbp in osteoclasts, we introduced Cbp into osteoclasts using an adenovirus gene delivery system. Introduction of Cbp stimulated recruitment of Csk into lipid rafts and suppressed c‐Src activity in a dose‐dependent manner. Furthermore, introduction of Cbp markedly inhibited formation of actin rings and bone‐resorbing activity in osteoclasts. In addition, treatment with RANKL and overexpression of TRAF6 or NFAT2 inhibited Cbp expression in the osteoclastogenic cell line RAW264.7 along with osteoclastic differentiation. NFAT2 overexpression also inhibited Cbp expression in spleen macrophages. Collectively, our results indicate that reduction in Cbp expression is responsible for maintaining high c‐Src activity in osteoclasts. These findings contribute to an understanding of the unique regulatory system for c‐Src in osteoclasts. © 2010 American Society for Bone and Mineral Research     

Coordination of microtubules and the actin cytoskeleton is important in osteoclast function, but calcitonin disrupts sealing zones without affecting microtubule networks

Bone-resorbing osteoclasts form sealing zones and ruffled borders toward the bone surface. The sealing zone consists of a ring-like alignment of F-actin dots and surrounds the ruffled border, from which protons are secreted into the bone surface. Vacuolar-type proton ATPase (V-ATPase) in osteoclasts is a ruffled border-associated enzyme responsible for the proton secretion. We studied the interaction between microtubules and the actin cytoskeleton in osteoclasts. Confocal microscopic observation revealed that osteoclasts on glass coverslips, dentine slices and Osteologictrade mark discs formed the ring-like structure of F-actin dots, and microtubules overlapped the top of the F-actin dots. Osteoclasts cultured on dentine formed resorption pits within 48 h. The treatment of osteoclasts with cytochalasin D, an F-actin-depolymerizing reagent, induced perturbation of the microtubules in osteoclasts on glass and inhibited their pit-forming activity on dentine in a dose-dependent and reversible manner. Conversely, nocodazole, a microtubule-depolymerizing reagent, disrupted sealing zones and inhibited pit-forming activity of osteoclasts in a dose-dependent and reversible manner. V-ATPase showed a tendency to be localized inside sealing zones in osteoclasts. Treatment of osteoclasts with calcitonin induced both disruption of sealing zones and dispersion of V-ATPase to the whole area of the cytoplasm within 60 min. The microtubule networks in osteoclasts remained unchanged for 60 min even in the presence of calcitonin. These results suggest that coordination of the actin cytoskeleton and microtubules is important in the function of osteoclasts, but calcitonin selectively affects the actin cytoskeleton and induces the dispersion of V-ATPase without causing significant changes in the microtubules.     

S 12911-2 inhibits osteoclastic bone resorption in vitro.

The potential anti-osteoporotic activity of the strontium compound, S12911, was tested on osteoclast-like cells and on cultured fetal mouse long bones. From 1 mM Sr2+, S12911 reduced both basal and stimulated bone resorption by decreasing osteoclast activity and ruffled border formation. The aim of this study was to evaluate the effects of S 12911-2 on osteoclastic bone resorption using in vitro systems. Osteoclast-like cells, produced in vitro by co-culture of mouse bone marrow cells with primary osteoblasts, were allowed to settle on dentine slices, and the area of resorption pits formed after 48 h was measured using an image analysis system. S 12911-2, at a minimal active concentration of 1 mM Sr2+, significantly reduced pit formation by these cells (p < 0.05). Pretreatment of slices for 48 h with S 12911-2 (5 mM Sr2+) did not produce appreciable inhibition of resorption. Bone resorption in cultured fetal mouse long bones was assessed by measuring the release of pre-incorporated 45calcium. S 12911-2 inhibited resorption in control cultures (18.9%, p < or = 0.05) and in bones cultured with the active form of vitamin D3 [1,25(OH)2D3] (44.5%, p < or = 0.05). S 12911-2 had no effect on the number of osteoclasts observed histochemically in longitudinal sections prepared from fetal mouse long bones. Electron microscopy of mouse long bones treated with S 12911-2 (3 mM Sr2+) showed osteoclasts with clear zones facing the bone surface, but without well-developed ruffled borders; untreated bones contained osteoclasts with normal ruffled borders. These results indicate that S 12911-2 inhibits osteoclast activity. This effect is directly linked to the presence of strontium, is effective on basal and stimulated resorption, and involves a decrease in ruffled border formation by osteoclasts.