The gingipains from Porphyromonas gingivalis do not directly induce osteoclast differentiation in primary mouse bone marrow cultures.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis is a major aetiological agent in the development of periodontitis, the major clinical hallmark of which is bone resorption. The cysteine proteases (gingipains) produced by P. gingivalis have a critical role in the pathogenesis of the disease, and previous studies on whole bacteria have implicated these enzymes in osteoclastogenesis, a process which serves to upregulate bone resorption. The effects of the gingipains from P. gingivalis on osteoclast differentiation were investigated here to determine whether the enzymes directly contribute to osteoclastogenesis and thus to bone resorption. MATERIAL AND METHODS: The effects of the gingipains on osteoclast differentiation were investigated in primary mouse bone marrow cultures. The cultures harvested from C57BL6/J mice were incubated in the presence of parathyroid hormone, a known osteoclastogenic factor, or active/inactivated forms of three gingipains. Osteoclast differentiation was quantified by counting the number of multinucleated cells positive for tartrate-resistant acid phosphatase, an enzyme marker for these cells. RESULTS: After 10 days of culture, the gingipains, either active or inactive, failed to stimulate osteoclast differentiation in comparison to the parathyroid hormone. CONCLUSION: The data presented here demonstrate that the gingipains do not induce osteoclast differentiation in this system, indicating that the bacterium uses other mechanisms to induce bone loss.

Protease-activated receptors in the musculoskeletal system.

Protease-activated receptors (PARs) mediate cellular responses to a subset of extracellular proteases, including blood coagulation factors and proteases produced by inflammatory cells. Cells in bone, cartilage and muscle exhibit cell type-specific expression patterns and functional responses for the different PARs. Activators of PAR-1 include thrombin, and activators of PAR-2 include trypsin and tryptase; PARs-3 and -4 are also receptors for thrombin. Thrombin stimulates PAR-1-mediated proliferative responses in osteoblasts, chondrocytes and myoblasts, and in developing muscle, PAR-1 activation by thrombin appears to mediate activity-dependent polyneuronal synapse reduction. In bone, activation of PAR-2 leads to inhibition of osteoblast-mediated osteoclast differentiation induced by hormones or cytokines, and in muscle, PAR-2 activation leads to stimulation of myoblast proliferation. Although there is some evidence for a role for PARs expressed by cells of the musculoskeletal system at specific stages of development, their major role appears to be in protecting the tissues from the destructive effects of inflammation and promoting regeneration. This review discusses the regulation of cell function in the musculoskeletal system by receptor-mediated responses to proteases. Expression patterns of PARs, the circumstances in which PAR activators are likely to be present, functional responses of PAR activation, and responses to thrombin for which receptors have not yet been identified are considered.

Thrombin inhibits osteoclast differentiation through a non-proteolytic mechanism.

Thrombin stimulates expression of interleukin 6 and cyclooxygenase 2 by osteoblasts, both of which enhance osteoblast-mediated osteoclast differentiation by increasing the ratio of receptor activator of nuclear factor κB ligand (RANKL) expression to that of osteoprotegerin (OPG) in osteoblasts. We hypothesised that thrombin would also increase this ratio and thereby stimulate osteoclast differentiation in mixed cultures of osteoblastic cells and osteoclast precursors. In primary mouse osteoblasts, but not in bone marrow stromal cells, thrombin increased the ratio of RANKL to OPG expression. Thrombin inhibited differentiation of osteoclasts, defined as tartrate-resistant acid phosphatase (TRAP)-positive cells with three or more nuclei, in mouse bone marrow cultures treated with osteoclastogenic hormones; this effect was not mediated by the major thrombin receptor, protease-activated receptor 1, nor did it require thrombin's proteolytic activity. Thrombin also caused a decrease in the number of TRAP-positive cells with fewer than three nuclei. Thrombin (active or inactive) also inhibited osteoclast differentiation and bone resorption, respectively, in cultures of mouse spleen cells and human peripheral blood mononuclear cells induced to undergo osteoclastogenesis by treatment with RANKL and macrophage colony-stimulating factor. Osteoclast differentiation in spleen cells was inhibited when they were exposed to thrombin from days 0 to 3 or 3 to 5 of culture but not days 5 to 7 when most fusion occurred. Thrombin inhibited expression of RANK by spleen cells. These observations indicate that, although thrombin stimulates production of osteoclastogenic factors by osteoblastic cells, it inhibits the early stages of RANKL-induced osteoclast differentiation through a direct effect on osteoclast precursors that does not require thrombin's proteolytic activity.