Department of Periodontology, Nihon University School of Dentistry at Matsudo, Chiba 271-8587, Japan.
J Cell Biochem. 2012 Sep;113(9):2822-34. doi: 10.1002/jcb.24157.
Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is believed to be associated with aggressive periodontitis characterized by a rapid bone loss. A. actinomycetemcomitans lipopolysaccharide (LPS) has a similar structure to Escherichia coli LPS, and they are Toll-like receptor 4 agonists. Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. To investigate the effects of A. actinomycetemcomitans LPS on bone formation, we targeted BSP as a marker for osteogenic differentiation and bone formation. BSP mRNA levels were decreased by 0.1 µg/ml and increased by 0.01 µg/ml A. actinomycetemcomitans LPS at 6 h in osteoblast-like ROS17/2.8 cells. In transient transfection analyses, 0.1 µg/ml decreased and 0.01 µg/ml A. actinomycetemcomitans LPS increased luciferase activities of the construct (-116 to +60). Introduction of 2 bp mutations to the constructs showed that the effects of A. actinomycetemcomitans LPS were mediated by a cAMP response element (CRE), a FGF2 response element (FRE), and a homeodomain protein-binding site (HOX). Tyrosine kinase, ERK1/2, and PI3-kinase/Akt participated in the effects of both 0.1 and 0.01 µg/ml A. actinomycetemcomitans LPS. The results of gel shift showed that 0.1 µg/ml decreased while 0.01 µg/ml A. actinomycetemcomitans LPS increased CRE-, FRE-, and HOX-binding protein complexes formation at 6 h, and revealed that 0.01 µg/ml A. actinomycetemcomitans LPS induced BSP transcription through CREB1, JunD, Fra2, c-Fos, Runx2, Dlx5, and Smad1 targeting those response elements. These studies therefore indicated that 0.1 µg/ml suppressed and 0.01 µg/ml A. actinomycetemcomitans LPS increased BSP gene transcription mediated through CRE, FRE, and HOX elements in the rat BSP gene promoter.
伴放线放线杆菌（A. actinomycetemcomitans）被认为与侵袭性牙周炎有关，其特征为快速骨丢失。A. actinomycetemcomitans 脂多糖（LPS）与大肠杆菌 LPS 结构相似，是 Toll 样受体 4 的激动剂。骨唾液蛋白（BSP）是成骨细胞分化的早期标志物。为了研究 A. actinomycetemcomitans LPS 对骨形成的影响，我们以 BSP 作为成骨分化和骨形成的标志物。在成骨样 ROS17/2.8 细胞中，0.1μg/ml A. actinomycetemcomitans LPS 在 6 小时降低 BSP mRNA 水平，而 0.01μg/ml A. actinomycetemcomitans LPS 则增加 BSP mRNA 水平。瞬时转染分析显示，0.1μg/ml A. actinomycetemcomitans LPS 降低构建体的荧光素酶活性（-116 至+60），而 0.01μg/ml A. actinomycetemcomitans LPS 则增加其活性。构建体中的 2 个碱基突变表明，A. actinomycetemcomitans LPS 的作用是通过 cAMP 反应元件（CRE）、FGF2 反应元件（FRE）和同源盒蛋白结合位点（HOX）介导的。酪氨酸激酶、ERK1/2 和 PI3-激酶/Akt 参与了 0.1μg/ml 和 0.01μg/ml A. actinomycetemcomitans LPS 的作用。凝胶迁移显示，0.1μg/ml A. actinomycetemcomitans LPS 在 6 小时时降低 CRE、FRE 和 HOX 结合蛋白复合物的形成，而 0.01μg/ml A. actinomycetemcomitans LPS 则增加其形成，表明 0.01μg/ml A. actinomycetemcomitans LPS 通过靶向这些反应元件的 CREB1、JunD、Fra2、c-Fos、Runx2、Dlx5 和 Smad1 诱导 BSP 转录。因此，这些研究表明，0.1μg/ml A. actinomycetemcomitans LPS 抑制，而 0.01μg/ml A. actinomycetemcomitans LPS 增加大鼠 BSP 基因启动子中 CRE、FRE 和 HOX 元件介导的 BSP 基因转录。
