Plasminogen activators are involved in the degradation of bone by osteoclasts

Osteoclastic bone degradation depends on the activity of several proteolytic enzymes, in particular to those belonging to the classes of cysteine proteinases and matrix metalloproteinases (MMPs). Yet, several findings suggest that the two types of plasminogen activators (PA), the tissue- and urokinase-type PA (tPA and uPA, respectively) are also involved in this process. To investigate the involvement of these enzymes in osteoclast-mediated bone matrix digestion, we analyzed bone explants of mice that were deficient for both tPA and uPA and compared them to wild type mice. The number of osteoclasts as well as their ultrastructural appearance was similar for both genotypes. Next, calvarial and metatarsal bone explants were cultured for 6 or 24 h in the presence of selective inhibitors of cysteine proteinases or MMPs and the effect on osteoclast-mediated bone matrix degradation was assessed. Inhibition of the activity of cysteine proteinases in explants of control mice resulted in massive areas of non-digested demineralized bone matrix adjacent to the ruffled border of osteoclasts, an effect already maximal after 6 h. However, at that time point these demineralized areas were not observed in bone explants from uPA/tPA deficient mice. After prolonged culturing (24 h), a comparable amount of demineralized bone matrix adjacent to actively resorbing osteoclasts was observed in the two genotypes, suggesting that degradation was delayed in uPA/tPA deficient bones. The activity of cysteine proteinases as assessed in bone extracts, proved to be higher in extracts from uPA/tPA(-/-) bones. Immunolocalization of the integrin alpha(v)beta(3) of in vitro generated osteoclasts demonstrated a more diffuse labeling of osteoclasts derived from uPA/tPA(-/-) mice. Taken together, our data indicate that the PAs play a hitherto unrecognized role in osteoclast-mediated bone digestion. The present findings suggest that the PAs are involved in the initial steps of bone degradation, probably by a proper integrin-dependent attachment to bone.     

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.     

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.     

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.     

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.     

Effects of the proteinase inhibitors leupeptin and E-64 on osteoclastic bone resorption

Summary To determine the possible involvement of cysteine-proteinases in bone matrix degradation by osteoclasts, the effects of the proteinase inhibitors leupeptin and E-64 were studied in anin vitro system using mouse bone explants. It was observed that in explants treated with the drugs, the amount of demineralized matrix opposing the ruffled border of the osteoclasts increased about 20-fold within 6 hours. This suggests that demineralization had proceeded whereas matrix degradation had been retarded. It was further noticed that in 12 of 287 osteoclasts, cytoplasmic vacuoles were present containing collagen fibrils that could not be distinguished from those in cartilage or bone. Their intracellular localization was proved by the study of serial sections. Finally, a significant reduction was shown as to the relative surface density of electrontranslucent vacuoles; this would seem to suggest reduced endocytic activity of the cells. Our observations support the view that cysteine-proteinases play an important role in osteoclastic bone resorption. It was further noticed that thein vitro effects of leupeptin and E-64 in certain respects resemble ultrastructural features of pycnodysostosis, an osteopetrosislike bone disorder. The data are in line with the hypothesis that this disease is caused by insufficient activity of osteoclastic cysteine-proteinases.     

Mechanism of osteoclastic bone resorption: A new hypothesis

Summary Osteoclastic bone resorption involves the solubilization of the mineral salts and the degradation of noncollagen bone matrix and collagen fibrils. As no recognizable collagen fibrils have ever been reported within cytoplasmic vacuoles in osteoclasts, it is generally assumed that the collagen fibrils are digested extracellularly in the resorption zone. The extent to which lysis occurs extracellularly and whether or not the osteoclasts phagocytose the degradation products remain to be established.In the present communication, a hypothesis is presented suggesting the possibility that osteoclastic resorption of bone involves the participation of two different cell types. According to this hypothesis, osteoclastic bone resorption is initiated by osteoclasts that demineralize areas of bone and degrade noncollagen bone matrix. After the osteoclasts have moved away or become partially detached from the demineralized site, the exposed collagen fibrils are phagocytosed by mononuclear, fibroblast-like or monocyte-derived cells.     

Human cystatin C, a cysteine proteinase inhibitor, inhibits bone resorption in vitro stimulated by parathyroid hormone and parathyroid hormone-related peptide of malignancy.

The effect of human recombinant cystatin C, a cysteine proteinase inhibitor, on bone resorption in vitro was evaluated. Bone resorption was assessed by analyzing the release of 45Ca and 3H from mouse calvarial bones prelabeled in vivo by injections with 45Ca or [3H]proline, respectively. In 24 h cultures, cystatin C (50 micrograms/ml) significantly inhibited the release of 45Ca and 3H stimulated by parathyroid hormone (PTH, 15 nmol/liter) or parathyroid hormone-related peptide of malignancy (PTHrP, 15 nmol/liter). The degree of inhibition caused by cystatin C in these 24 h cultures was similar to that caused by calcitonin (30 ng/ml). The inhibitory effect of cystatin C on 45Ca release induced by PTH was sustained in 96 h cultures, whereas the initial inhibition caused by calcitonin was transient. Cystatin C, 10-100 micrograms/ml, caused a dose-dependent inhibition of PTH (15 nmol/liter), and PTHrP (15 nmol/liter) stimulated 45Ca release. Addition of 50 micrograms/ml of cystatin C to mouse bone cultures inhibited the release of 45Ca induced by PTH and PTHrP at a wide range of submaximal and maximal concentrations of hormones (0.01-10 nmol/liter). No effect of cystatin C on 45Ca release in dead bones could be observed, nor did the inhibitor decrease the release of calcium in control bones. The inhibition by cystatin C on PTH-induced mineral mobilization was reversible. Cystatin C (1-100 micrograms/ml) did not affect protein synthesis or mitotic activities in mouse calvarial bones as assessed by the incorporation of [3H]proline and [3H]thymidine, respectively. These data show that cystatin C is a potent inhibitor of mineral mobilization and matrix degradation in cultured bones stimulated to resorb by PTH and PTHrP and that this effect is not due to general cytotoxicity.     

Quantitative analyses of osteoclast changes in resorbing bone organ cultures

Summary The stimulation of bone resorption, assessed by the release of⁴⁵Ca from prelabeled bones, was associated with an increase in number of osteoclasts per bone section in parathyroid hormone (PTH)-treated bones, but not in lipopolysaccharide (LPS)-treated bones. By contrast the number of nuclei per osteoclast increased following LPS treatment, but was not affected by PTH. LPS-treated bones had more multinucleated cells, some having as many as 27 nuclei per osteoclast. More osteoclasts were adjacent to the bone collar in bones treated with LPS or PTH than in control bones. In LPS-treated bones this area also contained the largest osteoclasts, as determined by the greatest number of nuclei per osteoclast. The results suggest that LPS and PTH stimulate osteoclastic resorption by different mechanisms.     

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.     

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 rapid appearance of acid phosphatase activity at the developing ruffled border of parathyroid hormone activated medullary bone osteoclasts

Summary The localization of acid phosphatase (ACPase) activity in and near parathyroid hormone (PTH) activated osteoclasts was investigated using electron microscopic cytochemical methods. At 3 hours after oviposition in Japanese Quail hens, medullary bone osteoclasts were highly reactive for ACPase but lacked ruffled borders. There was no evidence of extracellular ACPase activity associated with these osteoclasts. At 20 minutes after PTH administration, osteoclasts had developing ruffled borders and ACPase activity was found in the matrix and extracellular space adjacent to most of these ruffled borders. ACPase activity was seldom observed beyond the resorption zone delineated eated by the osteoclast clear zones. These results provide direct cytochemical evidence that the ruffled border functions in the release and/or activation of ACPase. In addition, these results show that ACPase localization is rapidly responsive to exogenous PTH.     

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.     

Ultrastructure, tartrate-resistant acid phosphatase activity and calcitonin responsiveness of osteoclasts at sites of demineralized bone matrix implant-induced osteogenesis

The morphology, ultrastructure, tartrate-resistance acid phosphatase reactivity, and calcitonin responsiveness of osteoclasts induced at sites of demineralized bone matrix (DBM) implant-induced osteogenesis in rats were determined. Osteoclasts at these ectopic sites had a morphologic and ultrastructural appearance similar to osteoclasts normally found in skeletal tissues. When observed by scanning electron microscopy, resorption surfaces on the implants had well-defined resorption pits (Howship's lacunae), indicative of active bone resorption. The osteoclasts stained intensely for tartrate-resistance acid phosphatase, an enzyme that is specific for osteoclasts. In response to human calcitonin, hypocalcemia occurred and osteoclasts lost their ruffled borders, indicating that these cells are responsive to exogenous hormonal stimulation. The osteoclasts induced by subcutaneous implantation of DBM had morphologic and functional characteristics similar to osteoclasts normally found in skeletal tissues.     

The type I collagen fragments ICTP and CTX reveal distinct enzymatic pathways of bone collagen degradation.

Bone resorption may generate collagen fragments such as ICTP and CTX, which can be quantified in serum and/or urine by using specific immunoassays, and which are used as clinical markers. However, the relative abundance of ICTP and CTX varies according to the type of bone pathology, suggesting that these two fragments are generated through distinct collagenolytic pathways. In this study, we analyzed the release of ICTP and CTX from bone collagen by the proteinases reported to play a role in the solubilization of bone matrix. Cathepsin K released large amounts of CTX, but did not allow a detectable release of ICTP. Conversely, the matrix metalloproteinases (MMPs) MMP-2, -9, -13, or -14 released ICTP, but did not allow a detectable release of CTX. Next we analyzed the release of ICTP and CTX from bone explants cultured in the presence of well-established inhibitors of these proteinases and of matrix solubilization. An inhibitor of cysteine proteinases including cathepsin K, inhibited the release of CTX, but not the release of ICTP. MMP inhibitors inhibited the release of ICTP, but also that of CTX, in agreement with the putative role of MMPs in the initiation of bone resorption in addition to matrix solubilization. Similarly the treatment of mice bearing bone metastasis with an MMP inhibitor led to a significant reduction of serum ICTP and CTX, and osteolytic lesions. We conclude that the generation of ICTP and CTX depends on different collagenolytic pathways. This finding may explain why these two markers may discriminate between different bone pathologies.     

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.     

Does collagen trigger the recruitment of osteoblasts into vacated bone resorption lacunae during bone remodeling?

Osteoblast recruitment during bone remodeling is obligatory to re-construct the bone resorbed by the osteoclast. This recruitment is believed to be triggered by osteoclast products and is therefore likely to start early during the remodeling cycle. Several osteoclast products with osteoblast recruitment potential are already known. Here we draw the attention on the osteoblast recruitment potential of the collagen that is freshly demineralized by the osteoclast. Our evidence is based on observations on adult human cancellous bone, combined with in vitro assays. First, freshly eroded surfaces where osteoblasts have to be recruited show the presence of non-degraded demineralized collagen and close cell–collagen interactions, as revealed by electron microscopy, while surface-bound collagen strongly attracts osteoblast lineage cells in a transmembrane migration assay. Compared with other extracellular matrix molecules, collagen's potency was superior and only equaled by fibronectin. Next, the majority of the newly recruited osteoblast lineage cells positioned immediately next to the osteoclasts exhibit uPARAP/Endo180, an endocytic collagen receptor reported to be involved in collagen internalization and cell migration in various cell types, and whose inactivation is reported to lead to lack of bone formation and skeletal deformities. In the present study, an antibody directed against this receptor inhibits collagen internalization in osteoblast lineage cells and decreases to some extent their migration to surface-bound collagen in the transmembrane migration assay. These complementary observations lead to a model where collagen demineralized by osteoclasts attracts surrounding osteoprogenitors onto eroded surfaces, and where the endocytic collagen receptor uPARAP/Endo180 contributes to this migration, probably together with other collagen receptors. This model fits recent knowledge on the position of osteoprogenitor cells immediately next to remodeling sites in adult human cancellous bone.     

Mechanisms of action of demineralized bone matrix in the repair of cortical bone defects

Demineralized bone matrix commonly is used to enhance and to facilitate bone grafting after skeletal injury or disease; however, the biologic bases for its bone-inducing abilities remain obscure. We have taken advantage of a mouse model of cortical bone defect healing to elucidate its mechanisms of action in vivo. Demineralized bone matrix combined with hyaluronan improved skeletal healing by inducing early deposition of an osteoid matrix. Demineralized bone matrix combined with hyaluronan might accelerate bone formation because it serves as a scaffold on which osteoprogenitor cells attach. We tested this possibility by comparing demineralized bone matrix combined with hyaluronan with heat-inactivated demineralized bone matrix combined with hyaluronan and found that the intact material was superior in terms of its ability to stimulate new bone formation. We also compared the bone inducing capacity of demineralized bone matrix combined with hyaluronan with a synthetic collagen sponge and found that not only the synthetic collagen scaffold delayed bone healing but also impaired bony bridging at later stages of repair. Another important property of demineralized bone matrix combined with hyaluronan was its ability to become actively degraded by osteoclasts during healing. Therefore, demineralized bone matrix combined with hyaluronan may not only attract osteoblasts and stimulate their differentiation, but also induce bone matrix resorption, which is a critically important regulator of bone formation and mineralization.     

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

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