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

牙龈卟啉单胞菌是牙周炎的主要病原体之一，以破骨细胞过度吸收牙槽骨为特征。这种细菌产生一种叫做gingipains的半胱氨酸蛋白酶，根据其裂解位点特异性分为赖氨酸特异性（Kgp）和精氨酸特异性牙龈蛋白酶（Rgps）。在这项研究中，我们研究了牙龈疼痛对破骨细胞分化的影响。在小鼠骨髓细胞和成骨细胞共培养中，Kgp能增强1α， 25-二羟基维生素D_3[1α， 25(OH)_2D_3]诱导的多核破骨细胞的形成，而RgpB则不能。生理浓度（0.1 nM）的1α， 25(OH)_2D_3在100 nM Kgp存在下诱导破骨细胞形成的程度与10 nM 1α， 25(OH)_2D_3诱导的程度相当。Kgp还能促进多种微生物成分（包括脂多糖）诱导的破骨细胞生成。Kgp与1α， 25(OH)_2D_3或脂多糖联合使用也增加了牙本质切片上形成的吸收坑的数量，表明Kgp存在下形成的破骨细胞具有骨吸收活性。Kgp增强的破骨细胞生成与共培养培养基中骨保护素的消耗和蛋白水解活性相关，因为Kgp抑制剂苯氧羰基-苯丙酰-酰基-无环基酮完全消除了Kgp诱导的破骨细胞生成。Kgp消化骨保护素，因为其重组蛋白在血清存在下易被Kgp降解。因此，Kgp不能促进骨保护素缺乏的成骨细胞和骨髓细胞的破骨细胞生成。此外，Kgp促进的破骨细胞生成被过量的重组骨保护素所消除。这些发现表明，骨保护素的降解是Kgp促进破骨细胞生成的机制之一。

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.