Okahashi Nobuo, Inaba Hiroaki, Nakagawa Ichiro, Yamamura Taihei, Kuboniwa Masae, Nakayama Koji, Hamada Shigeyuki, Amano Atsuo
Department of Oral Frontier Biology, Osaka University Graduate School of Dentistry, Osaka-Suita 565-0871, Japan.
Infect Immun. 2004 Mar;72(3):1706-14. doi: 10.1128/IAI.72.3.1706-1714.2004.
Porphyromonas gingivalis, an important periodontal pathogen, is closely associated with inflammatory alveolar bone resorption, and several components of the organism such as lipopolysaccharides have been reported to stimulate production of cytokines that promote inflammatory bone destruction. We investigated the effect of infection with viable P. gingivalis on cytokine production by osteoblasts. Reverse transcription-PCR and real-time PCR analyses revealed that infection with P. gingivalis induced receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL) mRNA expression in mouse primary osteoblasts. Production of interleukin-6 was also stimulated; however, osteoprotegerin was not. SB20350 (an inhibitor of p38 mitogen-activated protein kinase), PD98059 (an inhibitor of classic mitogen-activated protein kinase kinase, MEK1/2), wortmannin (an inhibitor of phosphatidylinositol 3 kinase), and carbobenzoxyl-leucinyl-leucinyl-leucinal (an inhibitor of NF-kappaB) did not prevent the RANKL expression induced by P. gingivalis. Degradation of inhibitor of NF-kappaB-alpha was not detectable; however, curcumin, an inhibitor of activator protein 1 (AP-1), prevented the RANKL production induced by P. gingivalis infection. Western blot analysis revealed that phosphorylation of c-Jun, a component of AP-1, occurred in the infected cells, and an analysis of c-Fos binding to an oligonucleotide containing an AP-1 consensus site also demonstrated AP-1 activation in infected osteoblasts. Infection with P. gingivalis KDP136, an isogenic deficient mutant of arginine- and lysine-specific cysteine proteinases, did not stimulate RANKL production. These results suggest that P. gingivalis infection induces RANKL expression in osteoblasts through AP-1 signaling pathways and cysteine proteases of the organism are involved in RANKL production.
牙龈卟啉单胞菌是一种重要的牙周病原体，与炎症性牙槽骨吸收密切相关，据报道该菌的一些成分如脂多糖可刺激细胞因子的产生，进而促进炎症性骨破坏。我们研究了活的牙龈卟啉单胞菌感染对成骨细胞细胞因子产生的影响。逆转录-聚合酶链反应（RT-PCR）和实时PCR分析显示，牙龈卟啉单胞菌感染可诱导小鼠原代成骨细胞中核因子κB受体激活剂配体（RANKL）mRNA表达。白细胞介素-6的产生也受到刺激；然而，骨保护素却未受影响。SB20350（p38丝裂原活化蛋白激酶抑制剂）、PD98059（经典丝裂原活化蛋白激酶激酶MEK1/2抑制剂）、渥曼青霉素（磷脂酰肌醇3激酶抑制剂）和苄氧羰基-亮氨酰-亮氨酰-亮氨酸（NF-κB抑制剂）均不能阻止牙龈卟啉单胞菌诱导的RANKL表达。未检测到NF-κB-α抑制剂的降解；然而，激活蛋白1（AP-1）抑制剂姜黄素可阻止牙龈卟啉单胞菌感染诱导的RANKL产生。蛋白质印迹分析显示，感染细胞中AP-1的组成成分c-Jun发生了磷酸化，对c-Fos与含有AP-1共有位点的寡核苷酸结合的分析也表明感染的成骨细胞中AP-1被激活。用精氨酸和赖氨酸特异性半胱氨酸蛋白酶的同基因缺陷突变体牙龈卟啉单胞菌KDP136感染不会刺激RANKL产生。这些结果表明，牙龈卟啉单胞菌感染通过AP-1信号通路诱导成骨细胞中RANKL表达，且该菌的半胱氨酸蛋白酶参与了RANKL的产生。
