A study on the mechanism of bone resorption by gingipains, the major proteases produced by Porphyromonas gingivalis

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Porphyromonas gingivalis is one of the major pathogens of periodontitis, a condition characterized by excessive alveolar bone resorption by osteoclasts. The bacterium produces cysteine proteases called gingipains, which are classified according to their cleavage-site specificity into lysine-specific(Kgp) and arginine-specific gingipains(Rgps). In this study, we examined the effects of gingipains on osteoclast differentiation. In co-cultures of mouse bone marrow cells and osteoblasts, formation of multinucleated osteoclasts induced by 1α, 25-dihydroxyvitamin D_3[1α, 25(OH)_2D_3] was augmented by Kgp but not by RgpB. A physiological concentration(0.1 nM) of 1α, 25(OH)_2D_3 induced the osteoclast formation in the presence of 100 nM Kgp to the extent comparable to that induced by 10 nM 1α, 25(OH)_2D_3. Kgp also enhanced osteoclastogenesis induced by various microbial components including lipopolysaccharide. Combined use of Kgp and 1α, 25(OH)_2D_3 or lipopolysaccharide also increased the number of resorption pits developed on dentin slices, indicating the osteoclasts formed in the presence of Kgp possess bone-resorbing activity. The enhanced osteoclastogenesis by Kgp was correlated with a depletion of osteoprotegerin in co-culture media and proteolytic activity-dependent, since benzyloxycarbonyl-phenylalanyl-lysyl-acycloxyketone, an inhibitor of Kgp, completely abolished osteoclastogenesis induced by Kgp. Kgp digested osteoprotegerin, since its recombinant protein was susceptible to degradation by Kgp in the presence of serum. As a result, Kgp did not augment osteoclastogenesis in co-cultures of osteoprotegerin-deficient osteoblasts and bone marrow cells. In addition, enhanced osteoclastogenesis by Kgp was abolished by excess amount of recombinant osteoprotegerin. These findings suggest that degradation of osteoprotegerin is one of the mechanisms underlying promotion of osteoclastogenesis by Kgp.