Yasuhara Rika, Miyamoto Yoichi, Takami Masamichi, Imamura Takahisa, Potempa Jan, Yoshimura Kentaro, Kamijo Ryutaro
Department of Biochemistry, Showa University School of Dentistry, Tokyo 142-8555, Japan.
Biochem J. 2009 Apr 1;419(1):159-66. doi: 10.1042/BJ20081469.
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.
牙龈卟啉单胞菌是牙周炎的主要致病菌之一，牙周炎的特征是破骨细胞导致牙槽骨过度吸收。该细菌产生一种名为牙龈蛋白酶的半胱氨酸蛋白酶，根据其切割位点特异性可分为Kgp（赖氨酸特异性牙龈蛋白酶）和Rgp（精氨酸特异性牙龈蛋白酶）。在本研究中，我们检测了牙龈蛋白酶对破骨细胞分化的影响。在小鼠骨髓细胞与成骨细胞的共培养中，1α,25(OH)₂D₃（1α,25 - 二羟基维生素D₃）诱导形成的多核破骨细胞，Kgp可增强其形成，而RgpB则无此作用。在存在100 nM Kgp的情况下，生理浓度（0.1 nM）的1α,25(OH)₂D₃诱导破骨细胞形成的程度与10 nM 1α,25(OH)₂D₃诱导的程度相当。Kgp还增强了包括脂多糖在内的各种微生物成分诱导的破骨细胞生成。Kgp与1α,25(OH)₂D₃或脂多糖联合使用也增加了牙本质切片上形成的吸收陷窝数量，表明在Kgp存在下形成的破骨细胞具有骨吸收活性。Kgp增强破骨细胞生成与共培养基中骨保护素的消耗相关，且依赖于蛋白水解活性，因为Kgp抑制剂苄氧羰基 - L - 苯丙氨酰 - L - 赖氨酰环氧化酮完全消除了Kgp诱导的破骨细胞生成。Kgp可消化骨保护素，因为其重组蛋白在血清存在下易被Kgp降解。因此，在骨保护素缺陷的成骨细胞与骨髓细胞的共培养中，Kgp并未增强破骨细胞生成。此外，过量的重组骨保护素消除了Kgp增强的破骨细胞生成。这些发现表明，骨保护素的降解是Kgp促进破骨细胞生成的潜在机制之一。
