Department of Periodontology, Nihon University School of Dentistry at Matsudo, Chiba 271-8587, Japan.
J Cell Biochem. 2010 Jul 1;110(4):823-33. doi: 10.1002/jcb.22594.
Lipopolysaccharide (LPS) is a major mediator of inflammatory response. Periodontopathic bacterium Porphyromonas gingivalis LPS has quite different character from Escherichia coli LPS. E. coli LPS is agonist for Toll-like receptor 4 (TLR4), whereas P. gingivalis LPS worked as antagonist for TLR4. Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. To investigate the effects of P. gingivalis LPS on BSP transcription, we used rat osteoblast-like ROS17/2.8 cells. BSP mRNA levels were decreased by 0.1 microg/ml and increased by 0.01 microg/ml P. gingivalis LPS at 12 h. Results of luciferase assays showed that 0.1 microg/ml decreased and 0.01 microg/ml P. gingivalis LPS increased BSP transcription in -116 to +60 BSP construct. The effects of P. gingivalis LPS were abrogated by double mutations in cAMP response element (CRE) and FGF2 response element (FRE). Tyrosine kinase inhibitor herbimycin A, ERK1/2 inhibitor and antioxidant N-acetylcystein inhibited effects of P. gingivalis LPS. Protein kinase A inhibitor and PI3-kinase/Akt inhibitor only abolished the effect of 0.01 microg/ml P. gingivalis LPS. Furthermore, 0.1 microg/ml LPS decreased the CRE- and FRE-protein complexes formation, whereas 0.01 microg/ml P. gingivalis LPS increased the nuclear protein binding to CRE and FRE. ChIP assays revealed increased binding of CREB1, JunD, Fra2, Runx2, Dlx5, and Smad1 to a chromatin fragment containing the CRE and FRE by 0.01 microg/ml P. gingivalis LPS. These studies therefore indicated that 0.1 microg/ml suppressed, and 0.01 microg/ml P. gingivalis LPS increased BSP gene transcription mediated through CRE and FRE elements in the rat BSP gene promoter.
脂多糖 (LPS) 是炎症反应的主要介质。牙周致病菌牙龈卟啉单胞菌 LPS 与大肠杆菌 LPS 有很大的不同。大肠杆菌 LPS 是 Toll 样受体 4 (TLR4) 的激动剂，而牙龈卟啉单胞菌 LPS 则作为 TLR4 的拮抗剂。骨唾液蛋白 (BSP) 是成骨细胞分化的早期标志物。为了研究牙龈卟啉单胞菌 LPS 对 BSP 转录的影响，我们使用大鼠成骨样 ROS17/2.8 细胞。0.1μg/ml 的牙龈卟啉单胞菌 LPS 在 12 小时使 BSP mRNA 水平降低，而 0.01μg/ml 的牙龈卟啉单胞菌 LPS 则增加。荧光素酶检测结果显示，0.1μg/ml 的牙龈卟啉单胞菌 LPS 降低了-116 至+60 BSP 构建体中的 BSP 转录，而 0.01μg/ml 的牙龈卟啉单胞菌 LPS 增加了 BSP 转录。cAMP 反应元件 (CRE) 和 FGF2 反应元件 (FRE) 的双突变消除了牙龈卟啉单胞菌 LPS 的作用。酪氨酸激酶抑制剂 herbimycin A、ERK1/2 抑制剂和抗氧化剂 N-乙酰半胱氨酸抑制了牙龈卟啉单胞菌 LPS 的作用。蛋白激酶 A 抑制剂和 PI3-激酶/Akt 抑制剂仅消除了 0.01μg/ml 牙龈卟啉单胞菌 LPS 的作用。此外，0.1μg/ml LPS 降低了 CRE 和 FRE-蛋白复合物的形成，而 0.01μg/ml 牙龈卟啉单胞菌 LPS 增加了核蛋白与 CRE 和 FRE 的结合。ChIP 检测显示，0.01μg/ml 牙龈卟啉单胞菌 LPS 增加了染色质片段中 CREB1、JunD、Fra2、Runx2、Dlx5 和 Smad1 与包含 CRE 和 FRE 的 CRE 和 FRE 结合。这些研究表明，0.1μg/ml 抑制了 0.01μg/ml 牙龈卟啉单胞菌 LPS 通过大鼠 BSP 基因启动子中的 CRE 和 FRE 元件增加 BSP 基因转录。
