Department of Periodontology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, Japan.
J Periodontal Res. 2010 Oct;45(5):602-11. doi: 10.1111/j.1600-0765.2010.01272.x. Epub 2010 Jun 10.
Amelogenins are hydrophobic proteins that are the major component of developing enamel. Enamel matrix derivative has been used for periodontal regeneration. Bone sialoprotein is an early phenotypic marker of osteoblast differentiation. In this study, we examined the ability of porcine amelogenins to regulate bone sialoprotein transcription.
To determine the molecular basis of the transcriptional regulation of the bone sialoprotein gene by amelogenins, we conducted northern hybridization, transient transfection analyses and gel mobility shift assays using the osteoblast-like ROS 17/2.8 cells.
Amelogenins (100 ng/mL) up-regulated bone sialoprotein mRNA at 3 h, with maximal mRNA expression occurring at 12 h (25 and 20 kDa) and 6 h (13 and 6 kDa). Amelogenins (100 ng/mL, 12 h) increased luciferase activities in pLUC3 (nucleotides -116 to +60), and 6 kDa amelogenin up-regulated pLUC4 (nucleotides -425 to +60) activity. The tyrosine kinase inhibitor inhibited amelogenin-induced luciferase activities, whereas the protein kinase A inhibitor abolished 25 kDa amelogenin-induced bone sialoprotein transcription. The effects of amelogenins were abrogated by 2-bp mutations in the fibroblast growth factor 2 response element (FRE). Gel-shift assays with radiolabeled FRE, homeodomain-protein binding site (HOX) and transforming growth factor-beta1 activation element (TAE) double-strand oligonucleotides revealed increased binding of nuclear proteins from amelogenin-stimulated ROS 17/2.8 cells at 3 h (25 and 13 kDa) and 6 h (20 and 6 kDa).
These results demonstrate that porcine 25 kDa amelogenin and its proteolytic derivatives stimulate bone sialoprotein transcription by targeting FRE, HOX and TAE in the bone sialoprotein gene promoter, and that full-length amelogenin and amelogenin cleavage products are able to regulate bone sialoprotein transcription via different signaling pathways.
釉原蛋白是疏水性蛋白，是发育釉质的主要成分。釉基质衍生物已被用于牙周组织再生。骨涎蛋白是成骨细胞分化的早期表型标志物。本研究旨在探讨猪釉原蛋白调节骨涎蛋白转录的能力。
为了确定釉原蛋白对骨涎蛋白基因转录调控的分子基础，我们使用成骨样 ROS 17/2.8 细胞进行了 northern 杂交、瞬时转染分析和凝胶迁移阻滞实验。
釉原蛋白（100ng/ml）在 3h 时上调骨涎蛋白 mRNA，12h 时达到最大表达（25 和 20kDa），6h 时达到最大表达（13 和 6kDa）。釉原蛋白（100ng/ml，12h）增加了 pLUC3（-116 至+60 核苷酸）的荧光素酶活性，6kDa 釉原蛋白增加了 pLUC4（-425 至+60 核苷酸）的活性。酪氨酸激酶抑制剂抑制了釉原蛋白诱导的荧光素酶活性，而蛋白激酶 A 抑制剂则消除了 25kDa 釉原蛋白诱导的骨涎蛋白转录。FRE 中的 2 个碱基对突变消除了釉原蛋白的作用。用放射性标记的 FRE、同源域蛋白结合位点（HOX）和转化生长因子-β1 激活元件（TAE）双链寡核苷酸进行凝胶阻滞实验显示，3h（25 和 13kDa）和 6h（20 和 6kDa）时，来自釉原蛋白刺激的 ROS 17/2.8 细胞的核蛋白结合增加。
这些结果表明，猪 25kDa 釉原蛋白及其蛋白水解衍生物通过靶向骨涎蛋白基因启动子中的 FRE、HOX 和 TAE 刺激骨涎蛋白转录，全长釉原蛋白和釉原蛋白裂解产物能够通过不同的信号通路调节骨涎蛋白转录。
