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 (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）是一种由分化的成骨细胞表达的矿化组织特异性蛋白，似乎在骨的初始矿化中起作用。本研究探讨了前列腺素E_2（PGE_2）和静磁场（SMF）对BSP基因表达的影响。为了探讨PGE_2和SMF对BSP表达的调控机制，我们分析了PGE_2和SMF对UMR 106细胞BSP表达的影响。实时荧光定量PCR检测PGE_2（3μM，12 h）和300和800 Gauss SMF（24 h）均能提高BSP mRNA的表达水平。对BSP启动子的进一步缺失分析表明，FGF反应元件（FRE）和cAMP反应元件（CRE）是PGE_2激活转录因子I的靶点，其作用可被蛋白激酶A、src酪氨酸激酶和MAP激酶抑制剂所抑制。PGE_2刺激后，核蛋白与放射性标记的FRE和CRE的结合增加，核蛋白与放射性标记的FRE和CRE的结合增加。为了进一步表征与CRE和FRE形成的复合物中的蛋白质，我们使用了几种转录因子的抗体。CREB抗体的加入破坏了CRE DNA-蛋白质复合物的形成，而细胞核提取物与抗磷酸化CREB抗体的孵育产生了可见的supershift复合物。在SMF刺激的UMR 106细胞核提取物中，核蛋白与放射性标记的FRE的结合增加，与Pit-1的结合减少，因此，这些研究确定了介导PGE_2和SMF诱导BSP转录的BSP基因近端启动子中的反应元件。

项目成果

山之内文彦 ほか: "リポポリサッカライド(LPS)による骨シアロタンパク質の転写の調節"日本歯科保存学雑誌. 46・3. 366-373 (2003)

Fumihiko Yamanouchi 等人：“脂多糖（LPS）对骨唾液蛋白转录的调节”日本保守牙科杂志 46·3（2003 年）。

E.Shimizu et al.: "Regulation of rat bone sialoprotein gene transcription by enamel matrix derivative."Journal of Periodontology. 75. 260-267 (2004)

E.Shimizu 等人：“牙釉质基质衍生物对大鼠骨唾液酸蛋白基因转录的调节”。牙周病学杂志。

E.Shimizu-Sasaki et al.: "Identification of FGF2-response element in the rat bone sialoprotein gene promoter."Connective Tissue Research. 44(Suppl.1). 103-108 (2003)

E.Shimizu-Sasaki 等人：“大鼠骨唾液蛋白基因启动子中 FGF2 反应元件的鉴定。”结缔组织研究。

Y.Ogataet et al.: "Tyrosine phosphorylation is involved in Ca^<2+> entry in human gingival fibroblasts."Cell Biology International. 27. 689-693 (2003)

Y.Ogataet等人：“酪氨酸磷酸化参与Ca 2+ 进入人牙龈成纤维细胞。”国际细胞生物学。

E.Shimizu et al.: "Prostaglandin E_2 stimulates bone sialoprotein (BSP) expression through cAMP and FGF2 response elements in the proximal promoter of the rat BSP gene."Journal of Biological Chemistry. 278. 28695-28667 (2003)

E.Shimizu 等人：“前列腺素 E_2 通过大鼠 BSP 基因近端启动子中的 cAMP 和 FGF2 反应元件刺激骨唾液蛋白 (BSP) 表达。”生物化学杂志。