Nukaga Jun, Kobayashi Makoto, Shinki Toshimasa, Song Hong, Takada Takatora, Takiguchi Takashi, Kamijo Ryutarou, Hasegawa Kohji
Department of Periodontology, Showa University Dental School, Tokyo, Japan.
J Periodontol. 2004 Feb;75(2):249-59. doi: 10.1902/jop.2004.75.2.249.
Receptor activator of nuclear factor-kappaB ligand (RANKL), which is expressed on the cell membrane of osteoblasts/stromal cells, stimulates osteoclastogenesis. We investigated the regulatory effects of interleukin-1beta (IL-1beta) and prostaglandin E2 (PGE2) on expression of RANKL in human periodontal ligament (HPDL) cells and the mechanisms involved in the PGE2 effect.
The HPDL cells were treated with IL-1beta, alone or in combination with indomethacin (INDO) or NS398, a cyclooxygenase-2 (COX-2) inhibitor. The HPDL cells were also pretreated with H89, a protein kinase A (PKA) inhibitor or GF109203X, a protein kinase C (PKC) inhibitor and subsequently treated with PGE2, PGE receptor (EP)2 agonist, EP4 agonist, forskolin, dibutyryl cAMP (db-cAMP), or 3-(isobutyl)-1-methylxantine (IBMX). After each treatment, expression of EP2, EP4, or RANKL mRNA was analyzed by reverse transcription-polymerase chain reaction and Southern hybridization. Expression of RANKL protein was detected by Western blotting, and cAMP accumulation was determined using a cAMP enzyme immunoassay kit.
IL-1beta stimulated the expression of RANKL at messenger RNA (mRNA) and protein levels in HPDL cells. Endogenous PGE2 partially mediated the IL-1beta-induced RANKL mRNA expression. Exogenously added PGE2 also stimulated RANKL expression at mRNA and protein levels in the cells. The PGE2-stimulated RANKL expression was mediated by EP2/4 and cAMP-dependent PKA, while PKC was possibly involved in the PGE2 action.
Human periodontal ligament cells activated with inflammatory factors such as IL-1beta and PGE2 may directly stimulate osteoclastogenesis through RANKL, which is stimulated to express by these factors.
核因子-κB 受体激活剂配体（RANKL）表达于成骨细胞/基质细胞的细胞膜上，可刺激破骨细胞生成。我们研究了白细胞介素-1β（IL-1β）和前列腺素 E2（PGE2）对人牙周膜（HPDL）细胞中 RANKL 表达的调节作用以及 PGE2 作用的相关机制。
HPDL 细胞单独用 IL-1β处理，或与吲哚美辛（INDO）或环氧合酶-2（COX-2）抑制剂 NS398 联合处理。HPDL 细胞还用蛋白激酶 A（PKA）抑制剂 H89 或蛋白激酶 C（PKC）抑制剂 GF109203X 预处理，随后用 PGE2、PGE 受体（EP）2 激动剂、EP4 激动剂、福斯可林、二丁酰环磷腺苷（db-cAMP）或 3-(异丁基)-1-甲基黄嘌呤（IBMX）处理。每次处理后，通过逆转录-聚合酶链反应和 Southern 杂交分析 EP2、EP4 或 RANKL mRNA 的表达。通过 Western 印迹检测 RANKL 蛋白的表达，并使用 cAMP 酶免疫分析试剂盒测定 cAMP 的积累。
IL-1β在 HPDL 细胞的信使核糖核酸（mRNA）和蛋白质水平上刺激 RANKL 的表达。内源性 PGE2 部分介导了 IL-1β诱导的 RANKL mRNA 表达。外源性添加的 PGE2 也在细胞的 mRNA 和蛋白质水平上刺激 RANKL 的表达。PGE2 刺激的 RANKL 表达由 EP2/4 和 cAMP 依赖性 PKA 介导，而 PKC 可能参与了 PGE2 的作用。
被 IL-1β和 PGE2 等炎性因子激活的人牙周膜细胞可能通过 RANKL 直接刺激破骨细胞生成，而这些因子可刺激 RANKL 表达。
