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.

Endocytosis of tartrate-resistant acid phosphatase by osteoblast-like cells is followed by inactivation of the enzyme.

Tartrate-resistant acid phosphatase (TRACP) is generally used as a marker of osteoclasts. Yet, other bone-associated cells, such as osteoblasts and osteocytes, may also express activity of this enzyme. Osteoblasts containing TRACP activity are seen particularly in the vicinity of areas of bone resorption, suggesting that osteoclasts somehow induce TRACP activity in osteoblasts. In a recent study, we found that osteoblast-like cells appeared to have the capacity to endocytose TRACP released by osteoclast precursors. In the present study, we investigated the endocytosis of TRACP in more detail as well as the fate of the endocytosed enzyme. We found that incubation of osteoblast-like cells with TRACP-coated beads resulted in attachment of a high number of beads to the cells. After culturing osteoblast-like cells with medium conditioned by blood monocytes that contain TRACP, activity of the enzyme was found in the cells. Following replacement of the medium by normal medium that did not contain TRACP, a decrease in the level of TRACP activity in osteoblast-like cells occurred. Our data strongly suggest that osteoblast-like cells recognize TRACP released by osteoclast precursors and that upon endocytosis inactivation of the enzyme occurs. We propose that uptake of the enzyme is important for the control of enzyme activity, thereby preventing degradation of matrix constituents.

Endogenous expression and endocytosis of tartrate-resistant acid phosphatase (TRACP) by osteoblast-like cells.

Tartrate-resistant acid phosphatase (TRACP) is produced by macrophages and other cells of the monohistiocytic lineage. In particular, osteoclasts are characterized for a high expression of this enzyme. Yet, several data suggest that other bone cell types, such as osteocytes and osteoblasts, may also express activity of this enzyme. This is particularly obvious at sites were osteoclasts resorb bone, suggesting that osteoclasts (or their precursors) somehow induce TRACP activity in osteoblasts. In the present study, we investigated this by culturing human osteoblast-like cells with and without conditioned medium (MCM) from human blood monocytes (as a source of osteoclast precursors). High levels of TRACP activity were found in osteoblast-like cells cultured with MCM. Depletion of TRACP from this medium resulted in the absence of its activity in osteoblast-like cells, thus suggesting that the TRACP activity in these cells was the result of endocytosed TRACP that was released by the monocytes in the MCM. Osteoblast-like cells cultured in control (non-conditioned) medium contained very low levels of TRACP-like activity. However, the cells expressed TRACP mRNA and incubation of extracts of these cells with active cathepsin B did induce activity of a TRACP-like enzyme. Inhibition of the activity of cysteine proteinases in general and of cathepsin B in particular, completely blocked TRACP activity of the osteoblast-like cells. This TRACP-like enzyme but not the alleged endocytosed fraction of TRACP was inhibited by fluoride, suggesting that the fractions may be different isoenzymes. Our data seem to indicate that osteoblast-like cells may contain two different fractions of TRACP, one that is released by monocytes and subsequently endocytosed by osteoblast-like cells and a second endogenous fraction that is present in an inactive proform. We hypothesize that the capacity of osteoblast-like cells to endocytose TRACP is important for the removal of this enzyme during or following the bone resorptive activity of the osteoclast.

Loss-of-function mutations in cathepsin C in two families with Papillon-Lefèvre syndrome are associated with deficiency of serine proteinases in PMNs.

Papillon-Lefèvre syndrome (PLS) is a rare autosomal recessive disease that involves severe periodontitis and hyperkeratosis of the hand palms and foot soles. Recently it was found that PLS patients carry loss-of-function mutations in the gene encoding cathepsin C (CTSC). In the present study we have analyzed the CTSC gene in two unrelated families with PLS. In the first non-consanguineous family, mutation analysis revealed the previously reported c.815G>C/p.R272P mutation. The second consanguineous family displayed a c.1213C>A mutation which resulted in the novel mutation p.H405N and is the first mutation described in the active site of the enzyme. The PLS patients had, next to the absence of cathepsin C activity in polymorphonuclear leukocytes (PMNs), no activity of the three serine proteinases elastase, cathepsin G and proteinase 3. Serine proteinases are supposed to be important in both the innate and adaptive immune systems. Their absence in PLS patients could explain the inadequate defense to periodontal infection.

Host-derived proteinases and degradation of dentine collagen in situ.

Dentine root caries is a process of demineralization and degradation of the organic matrix by proteinases. In this in situ study, the presence and activity of the matrix metalloproteinases 1, 2 and 9 (MMP-1, MMP-2, MMP-9) in saliva and in completely demineralized dentine specimens were investigated. Furthermore, the activity of cathepsin B was determined in saliva. A correlation between these enzymes and the level of degraded collagen was investigated. Demineralized dentine specimens were mounted in the partial prosthesis of 17 volunteers. Saliva samples were taken at 0, 2 and 4 weeks. After 4 weeks, the enzymes were extracted from the dentine specimens and the collagen loss was assessed. The collagen loss varied between 0 and 40.3%. Zymography of the saliva and the dentine extract samples showed that (pro-)MMP-2 and (pro-)MMP-9 were present. The levels of active MMPs were assessed, using fluorogenic MMP-specific substrates. All but 3 of the 51 saliva samples showed MMP-1 activity ranging from 1.5 to 101.1 relative fluorescence units (RFU)/s. Forty-eight saliva samples showed gelatinolytic MMP-2/MMP-9 activity (1.7-141.1 RFU/s). MMP-1 activity was shown in all dentine extracts varying between 3.5 and 295.0 RFU/s. From the dentine extracts, 15 showed MMP-2/MMP-9 activity (0.2-13.7 RFU/s). The MMP activity from both saliva and dentine extracts did not correlate with the collagen loss. The activity of salivary cathepsin B varied from 4.8 to 42.2 arbitrary units/min. A positive correlation was found between salivary MMP activity and cathepsin B activity. This study revealed that gelatinolytic enzyme activity was present both in saliva and dentine collagen. No correlation could be observed, however, between the level of enzyme activity and the collagen loss of the dentine specimens.

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

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

Functional heterogeneity of osteoclasts: matrix metalloproteinases participate in osteoclastic resorption of calvarial bone but not in resorption of long bone.

Data in the literature suggest that site-specific differences exist in the skeleton with respect to digestion of bone by osteoclasts. Therefore, we investigated whether bone resorption by calvarial osteoclasts (intramembranous bone) differs from resorption by long bone osteoclasts (endochondral bone). The involvement of two major classes of proteolytic enzymes, the cysteine proteinases (CPs) and matrix metalloproteinases (MMPs), was studied by analyzing the effects of selective low molecular weight inhibitors of these enzymes on bone resorption. Mouse tissue explants (calvariae and long bones) as well as rabbit osteoclasts, which had been isolated from both skeletal sites and subsequently seeded on bone slices, were cultured in the presence of inhibitors and resorption was analyzed. The activity of the CP cathepsins B and K and of MMPs was determined biochemically (CPs and MMPs) and enzyme histochemically (CPs) in explants and isolated osteoclasts. We show that osteoclastic resorption of calvarial bone depends on activity of both CPs and MMPs, whereas long bone resorption depends on CPs, but not on the activity of MMPs. Furthermore, significantly higher levels of cathepsin B and cathepsin K activities were expressed by long bone osteoclasts than by calvarial osteoclasts. Resorption of slices of bovine skull or cortical bone by osteoclasts isolated from long bones was not affected by MMP inhibitors, whereas resorption by calvarial osteoclasts was inhibited. Inhibition of CP activity affected the resorption by the two populations of osteoclasts in a similar way. We conclude that this is the first report to show that significant differences exist between osteoclasts of calvariae and long bones with respect to their bone resorbing activities. Resorption by calvarial osteoclasts depends on the activity of CPs and MMPs, whereas resorption by long bone osteoclasts depends primarily on the activity of CPs. We hypothesize that functionally different subpopulations of osteoclasts, such as those described here, originate from different sets of progenitors.

Participation of intracellular cysteine proteinases, in particular cathepsin B, in degradation of collagen in periosteal tissue explants.

The involvement of cysteine proteinases in the degradation of soft connective tissue collagen was studied in cultured periosteal explants. Using cysteine proteinase inhibitors that were active intracellularly or extracellularly (Ep453 and Ep475, respectively), it was shown that over-all collagen degradation, as measured by the release of hydroxyproline, decreased significantly on inhibition of the intracellular pool of cysteine proteinases by Ep453. This inhibitor also induced an accumulation of intracellular fibrillar collagen in fibroblasts, indicating a decreased degradation of phagocytosed collagen. The extracellular inhibitor, Ep475, had minor or no effects. Histochemical analysis using a substrate for the cysteine proteinases cathepsins B and L revealed a high level of enzyme activity, which was completely blocked in explants preincubated with a selective intracellular inhibitor of cathepsin B, Ca074-Me. Moreover, the cathepsin B inhibitor strongly affected collagen degradation, decreasing the release of hydroxyproline and increasing the accumulation of phagocytosed collagen. These effects were comparable or slightly stronger than those found with the general intracellular inhibitor (Ep453). Taken together, these data strongly suggest that intracellular cysteine proteinases, in particular cathepsin B, play an important role in the digestion of soft connective tissue collagen.

The identification of type 1 Gaucher disease patients, asymptomatic cases and carriers in The Netherlands using urine samples: an evaluation.

The feasibility of using urine samples for the identification of patients with Gaucher disease and carriers has been investigated. It was found that the pH of a urine sample should be pH 6.0 or lower to ensure stability of lysosomal hydrolases. Two parameters of glucocerebrosidase, which is deficient in Gaucher disease, were studied using urine samples from control subjects, obligate carriers and patients. Firstly, the relative level of glucocerebrosidase activity was measured by relating the activity of the enzyme to that of another lysosomal hydrolase. Secondly, the enzymic activity of glucocerebrosidase per unit of protein was measured using an immunological method. The first method allowed discrimination of nearly all obligate carriers of type 1 Gaucher disease from normal individuals. The second method allowed clear discrimination of the majority of carriers from normal individuals, but some obligate carriers were not distinguishable from normal subjects on the basis of this parameter. However, the combination of both methods allowed discrimination between all obligate carriers examined so far (n = 34) and controls (n = 86). There was variability between healthy individuals in the relative amount of glucocerebrosidase in urine samples. A small proportion of healthy individuals have a relatively high activity of glucocerebrosidase in urine samples, reminiscent of observations made in white blood cells by other investigators. In urine samples from two unrelated parents of Gaucher disease patients a level of glucocerebrosidase activity was present that could not be distinguished from that in samples of patients. These individuals represent cases with subclinical manifestation of Gaucher disease, illustrating once more the remarkable heterogeneity in clinical expression of this disorder.