Fujihara R, Usui M, Yamamoto G, Nishii K, Tsukamoto Y, Okamatsu Y, Sato T, Asou Y, Nakashima K, Yamamoto M
Department of Periodontology, Showa University School of Dentistry, Tokyo, Japan.
J Periodontal Res. 2014 Aug;49(4):508-17. doi: 10.1111/jre.12131. Epub 2013 Sep 19.
Periodontitis is an inflammatory disorder of the supporting tissue of teeth, which is composed of gingival soft tissue, cementum covering the tooth root, alveolar bone and periodontal ligament. The receptor activator of nuclear factor kappa B ligand (RANKL) is known to be an essential factor for osteoclastogenesis. Recent clinical studies indicate that levels of RANKL in the gingival crevicular fluid are increased while levels of its decoy receptor, osteoprotegerin (OPG), are decreased in patients with periodontitis. Although the gingival sulcus is composed of gingival tissue, RANKL and OPG expression in gingival epithelial cells is not fully understood. The aim of this study is to investigate the expression of RANKL and OPG in gingival tissue and which factors regulate RANKL expression in gingival epithelial cells.
Reverse transcriptase polymerase chain reaction analysis, western blotting and immunohistochemistry were performed to confirm RANKL and OPG expression in gingival epithelial cells (GECs) and in gingival tissue. Immunostaining was also examined to confirm tumor necrosis factor (TNF)-α and TNF receptor type 1 (TNFR1) expression in gingival tissue. Ca9-22 cells, a human gingival epithelial cell line and human primary GECs were treated with TNF-α. Ca9-22 cells were treated by antibodies against TNF receptors, an inhibitor and an activator of protein kinase A (PKA) signaling and inhibitors of p38, Erk and NF-κB signaling to examine TNF-α-RANKL signaling pathways.
RANKL mRNA and protein were expressed in GECs. Immunohistochemistry also showed RANKL expression in gingival tissue. On the other hand, the reverse transcriptase polymerase chain reaction and immunohistochemistry assay showed that GECs did not express OPG. In addition, TNF-α and TNFR1 proteins were expressed in junctional epithelium. TNF-α increased RANKL expression in GECs. TNF-α-induced RANKL expression was inhibited by an antibody against TNFR1 and an inhibitor of PKA signaling. Surprisingly, forskolin, a PKA activator, increased TNF-α-induced RANKL expression.
RANKL, TNF and TNFR1 were coexpressed in junctional epithelium of gingival tissue. TNF-α induced RANKL expression via TNFR1 and PKA signaling in GECs of junctional epithelium.
牙周炎是牙齿支持组织的一种炎症性疾病，牙齿支持组织由牙龈软组织、覆盖牙根的牙骨质、牙槽骨和牙周韧带组成。已知核因子κB受体活化因子配体（RANKL）是破骨细胞生成的关键因子。最近的临床研究表明，牙周炎患者龈沟液中RANKL水平升高，而其诱饵受体骨保护素（OPG）水平降低。虽然龈沟由牙龈组织构成，但牙龈上皮细胞中RANKL和OPG的表达尚未完全明确。本研究旨在调查牙龈组织中RANKL和OPG的表达情况以及哪些因素调节牙龈上皮细胞中RANKL的表达。
采用逆转录聚合酶链反应分析、蛋白质印迹法和免疫组织化学法，以确认RANKL和OPG在牙龈上皮细胞（GECs）和牙龈组织中的表达。还进行免疫染色以确认牙龈组织中肿瘤坏死因子（TNF）-α和肿瘤坏死因子受体1型（TNFR1）的表达。用人牙龈上皮细胞系Ca9-22细胞和人原代GECs分别用TNF-α处理。用抗TNF受体抗体、蛋白激酶A（PKA）信号通路的抑制剂和激活剂以及p38、Erk和NF-κB信号通路的抑制剂处理Ca9-22细胞，以检测TNF-α-RANKL信号通路。
GECs中表达RANKL mRNA和蛋白。免疫组织化学也显示牙龈组织中有RANKL表达。另一方面，逆转录聚合酶链反应和免疫组织化学分析显示GECs不表达OPG。此外，TNF-α和TNFR1蛋白在结合上皮中表达。TNF-α增加了GECs中RANKL的表达。抗TNFR1抗体和PKA信号通路抑制剂可抑制TNF-α诱导的RANKL表达。令人惊讶的是，PKA激活剂福司可林增加了TNF-α诱导的RANKL表达。
RANKL、TNF和TNFR1在牙龈组织的结合上皮中共表达。TNF-α通过TNFR1和PKA信号通路诱导结合上皮GECs中RANKL的表达。
