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.

Alpha11 beta1 integrin-dependent regulation of periodontal ligament function in the erupting mouse incisor.

The fibroblast integrin alpha11beta1 is a key receptor for fibrillar collagens. To study the potential function of alpha11 in vivo, we generated a null allele of the alpha11 gene. Integrin alpha11(-/-) mice are viable and fertile but display dwarfism with increased mortality, most probably due to severely defective incisors. Mutant incisors are characterized by disorganized periodontal ligaments, whereas molar ligaments appear normal. The primary defect in the incisor ligament leads to halted tooth eruption. alpha11beta1-defective embryonic fibroblasts displayed severe defects in vitro, characterized by (i) greatly reduced cell adhesion and spreading on collagen I, (ii) reduced ability to retract collagen lattices, and (iii) reduced cell proliferation. Analysis of matrix metalloproteinase in vitro and in vivo revealed disturbed MMP13 and MMP14 synthesis in alpha11(-/-) cells. We show that alpha11beta1 is the major receptor for collagen I on mouse embryonic fibroblasts and suggest that alpha11beta1 integrin is specifically required on periodontal ligament fibroblasts for cell migration and collagen reorganization to help generate the forces needed for axial tooth movement. Our data show a unique role for alpha11beta1 integrin during tooth eruption.

Role of polymorphonuclear leukocyte-derived serine proteinases in defense against Actinobacillus actinomycetemcomitans.

Periodontitis is a chronic destructive infection of the tooth-supportive tissues, which is caused by pathogenic bacteria such as Actinobacillus actinomycetemcomitans. A severe form of periodontitis is found in Papillon-Lefèvre syndrome (PLS), an inheritable disease caused by loss-of-function mutations in the cathepsin C gene. Recently, we demonstrated that these patients lack the activity of the polymorphonuclear leukocyte (PMN)-derived serine proteinases elastase, cathepsin G, and proteinase 3. In the present study we identified possible pathways along which serine proteinases may be involved in the defense against A. actinomycetemcomitans. Serine proteinases are capable to convert the PMN-derived hCAP-18 into LL-37, an antimicrobial peptide with activity against A. actinomycetemcomitans. We found that the PMNs of PLS patients released lower levels of LL-37. Furthermore, because of their deficiency in serine proteases, the PMNs of PLS patients were incapable of neutralizing the leukotoxin produced by this pathogen, which resulted in increased cell damage. Finally, the capacity of PMNs from PLS patients to kill A. actinomycetemcomitans in an anaerobic environment, such as that found in the periodontal pocket, seemed to be reduced. Our report demonstrates a mechanism that suggests a direct link between an inheritable defect in PMN functioning and difficulty in coping with a periodontitis-associated pathogen.

Osteoclastic bone degradation and the role of different cysteine proteinases and matrix metalloproteinases: differences between calvaria and long bone.

UNLABELLED: 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.

Structure of the periodontium in cathepsin C-deficient mice.

Papillon-Lefevre syndrome is characterized by increased susceptibility to early-onset periodontitis and is caused by mutations in the cathepsin C gene. How deficiency of the enzyme relates to an increased periodontal infection risk is still not entirely clear. One possibility is that the deficiency leads to changes in the structure of the periodontal tissues as a result of which its barrier function to pathogens is compromised. We studied the structure of the periodontium in 9-month-old cathepsin C-deficient mice (cathepsin C(-/-)) and compared this with age-matched wild-type mice. Our observations showed that the overall structure of the gingiva, periodontal ligament, alveolar process, and cementum layer are normal in cathepsin C(-/-) mice, with one exception, namely that epithelial rests of Malassez in the periodontal ligament of the cathepsin C(-/-) mice are slightly enlarged. In both experimental and control animals, we noted cyst formation in rests of Malassez. No signs of periodontal infection were observed. It is concluded that cathepsin C deficiency does not lead to major changes in the structure of the periodontium.

Gingival fibroblasts are better at inhibiting osteoclast formation than periodontal ligament fibroblasts.

Various studies indicate that periodontal ligament fibroblasts (PLF) have some similarities to osteoblasts, for example they have the capacity to induce the formation of osteoclast-like cells. Here, we investigated whether a second population of tooth-associated fibroblasts, gingival fibroblasts (GF), has similar osteoclastogenesis properties. PLF and GF were co-cultured with peripheral blood mononuclear cells (PBMC) in the presence and absence of dexamethasone and 1alpha,25dihydroxycholecalciferol (dex + vit D(3)) on plastic and on cortical bone slices. Tartrate resistant acid phosphatase (TRACP) positive multinucleated cells (MNCs) were more abundant in co-cultures with PLF than in GF-PBMC co-cultures, more abundant on plastic compared to bone and more abundant in the presence of dex + vit D(3). In line with these findings was an inhibition of MNC formation and not inhibition of existing osteoclasts by medium conditioned by GF. We next investigated whether expression of molecules important for osteoclastogenesis differed between the two types of fibroblasts and whether these molecules were regulated by dex + vit D(3). OPG was detected at high levels in both fibroblast cultures, whereas RANKL could not be detected. Resorption of bone did not occur by the MNCs formed in the presence of either fibroblast subpopulation, suggesting that the fibroblasts secrete inhibitors of bone resorption or that the osteoclast-like cells were not functional. The incapacity of the MNCs to resorb was abolished by culturing the fibroblast-PBMC cultures with M-CSF and RANKL. Our results suggest that tooth-associated fibroblasts may trigger the formation of osteoclast-like cells, but more importantly, they play a role in preventing bone resorption, since additional stimuli are required for the formation of active osteoclasts.

Site-specific localization of two distinct phosphatases along the osteoblast plasma membrane: tissue non-specific alkaline phosphatase and plasma membrane calcium ATPase.

In osteoblasts, alkaline phosphatase has been reported to be restricted to the basolateral domains. In recent studies, we have demonstrated phosphatase activities different from those of tissue non-specific alkaline phosphatase (TNSALP) along the osteoidal aspect of osteoblast membrane at alkaline and neutral pH on undecalcified freshly frozen sections of rat bones. In the present study, we sought to further characterize and define the nature of membrane-associated phosphatases along the osteoidal aspect of osteoblasts. Histochemical properties of the enzymes and their localization in vivo were examined in long bones of normal Wistar rats and TNSALP null mutant mice and their wild type littermates. Molecular profiles of the enzymes in the osteoblast extracts were also examined. The enzymatic activity of the phosphatase along the osteoidal surface of osteoblasts proved to be activated by both Mg2+ and Ca2+. Unlike TNSALP, the activity was inhibited by vanadate but resistant to levamisole, implicating a similarity between this enzyme and plasma membrane Ca2+ transport ATPase (PMCA). Immunohistochemistry showed that PMCA immunoreactions were restricted to the osteoidal domain of the plasma membrane. Native-PAGE analysis of osteoblast extracts suggested the presence of two phosphatases corresponding, respectively, to TNSALP and PMCA. Western blot analysis after SDS-PAGE of osteoblast extracts confirmed the existence of PMCA (140 kDa) and TNSALP (80 kDa). Gel-chemical analysis of the osteoblast extract from TNSALP null mutant mice depicted phosphatase activity, which was resistant to levamisole. These data suggest the presence of a phosphatase different from TNSALP, most plausibly PMCA, on the osteoidal surface of osteoblasts.

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.