Lysine-specific gingipain promotes lipopolysaccharide- and active-vitamin D3-induced osteoclast differentiation by degrading osteoprotegerin

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 Kgps (lysine-specific gingipains) and Rgps (arginine-specific gingipains). In the present 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 1alpha,25(OH)(2)D(3) (1alpha,25-dihydroxyvitamin D(3)) was augmented by Kgp but not by RgpB. A physiological concentration (0.1 nM) of 1alpha,25(OH)(2)D(3) induced the osteoclast formation in the presence of 100 nM Kgp to an extent comparable with that induced by 10 nM 1alpha,25(OH)(2)D(3). Kgp also enhanced osteoclastogenesis induced by various microbial components, including lipopolysaccharide. Combined use of Kgp and 1alpha,25(OH)(2)D(3) or lipopolysaccharide also increased the number of resorption pits developed on dentin slices, indicating that 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 medium and was proteolytic-activity-dependent, since benzyloxycarbonyl-L-phenylalanyl-L-lysylacycloxyketone, 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 an excess amount of recombinant osteoprotegerin. These findings suggest that degradation of osteoprotegerin is one of the mechanisms underlying promotion of osteoclastogenesis by Kgp.