Change of thegene expression of bone sialoprotein and transcription factors in osteoblasts during periodontal tissue regeration.

牙周组织再生过程中成骨细胞骨唾液酸蛋白和转录因子基因表达的变化

• 批准号: 14571989
• 负责人: OGATA Yorimasa
• 金额: $ 1.54万
• 依托单位: Nihon University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571989/
• 关键词: Bone sialoprotein; Extracellular matrix; Regeneration; Transcription factor; Transcriptional regulation; Gene promoter; Calcification; Tissue specificity; 遺伝子プロモータ-; Bone sialoprotein; Extracellular matrix; Regeneration; Transcription factor; Transcriptional regulation; Gene promoter; Calcification; Tissue specificity; 遺伝子プロモータ-

Bone sialoprotein (BSP) is a mineralized tissue-specific protein expressed by differentiated osteoblasts that appears to function in the initial mineralization of bone. In this study, we investigated the effects of prostaglandin E_2 (PGE_2) and static magnetic fields (SMF) on BSP gene expression. To determine the molecular mechanism of PGE_2 and SMF regulation of BSP, we analyzed the effects of the PGE_2 and SMF on the expression of BSP in UMR 106 cells. PGE_2 (3μM, 12h) and application of 300 and 800 Gauss SMF (24h) increased BSP mRNA levels detected by real-time PCR.From transient transfection assays using various BSP promoter-luciferase constructs, PGE_2 and SMF increased expression of the construct (pLUC3 ; -116 to +60). Further deletion analysis of the BSP promoter showed that a FGF response element (FRE) and a cAMP response element (CRE) were identified as a target of transcriptional I activation by PGE_2, the effects of which were inihibited by protein kinase A, src tyrosine kinase and MAP kinase inhibitors. FRE and a pituitary-specific transcription factor-1 regulatory element (Pit-1) were identified as a target for SMF, the effect of which was inihibited by tyrosine kinase inhibitor.Binding of nuclear proteins to a radiolabeled FRE and CRE were increased after stimulation by PGE_2. To further characterize the proteins in the complexes formed with the CRE and FRE, we used antibodies for several transcription factors. The addition of antibody to CREB disrupted the formation of the CRE DNA-protein complexes, while incubation of nuclear extracts with anti-phospho-CREB antibody produced a visible supershift complex. Binding of nuclear proteins to a radiolabeled FRE was increased and that to a Pit-1 was decreased in nuclear extracts prepared from SMF-stimulated UMR 106 cells.These studies, therefore, have identified response elements in the proximal promoter of the BSP gene that mediates PGE_2 and SMF-induced BSP transcription.

骨唾液蛋白（BSP）是一种矿化组织特异性蛋白，该蛋白是由分化成骨细胞表达的，似乎在骨的初始矿化中起作用。在这项研究中，我们研究了前列腺素E_2（PGE_2）和静态磁场（SMF）对BSP基因表达的影响。为了确定BSP的PGE_2和SMF调控的分子机制，我们分析了PGE_2和SMF对UMR 106细胞中BSP表达的影响。 PGE_2（3μm，12h）以及300和800高斯SMF（24H）的应用增加了通过实时PCR检测到的BSP mRNA水平。从瞬时翻译分析使用各种BSP启动子 - 荧光酶型构建体，PGE_2和SMF，PGE_2和SMF增加了构建体的表达（PLUC3; -116至+60）。 BSP启动子的进一步缺失分析表明，FGF响应元件（FRY）和CAMP响应元件（CRE）被识别为PGE_2的转录I激活的靶标，其作用是蛋白激酶A，SRC酪氨酸激酶和MAP激酶抑制剂的影响。 FRE和Pitutai特异性转录因子-1调节元（PIT-1）被确定为SMF的靶标，酪氨酸激酶抑制剂的效果不受抑制作用。核蛋白在PGE_2刺激后增加了放射性标记的FRE和CRE。为了进一步表征由CRE和FRE形成的络合物中的蛋白质，我们使用抗体用于几种转录因子。添加抗体以破坏了CRE DNA-蛋白质复合物的形成，而核提取物用抗磷酸-CREB抗体孵育产生了可见的超递变复合物。核蛋白与放射标记的FRE的结合增加了，并且在SMF刺激的UMR 106细胞中制备的核提取物中降低了对PIT-1的结合。