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

骨涎蛋白(BSP)是一种矿化的组织特异性蛋白，由分化的成骨细胞表达，似乎在骨的初始矿化过程中发挥作用。本研究探讨了前列腺素E_2(PGE_2)和恒磁场(SMF)对BSP基因表达的影响。为探讨前列腺素E_2(PGE_2)和SMF对BSP调控的分子机制，我们分析了PGE_2和SMF对UMR 106细胞BSP表达的影响。PGE_2(3μM，12h)和300和800Gauss SMF作用(24小时)可增加实时荧光定量PCR检测到的BSP基因表达水平。通过瞬时转染法检测，PGE_2和SMF可增加该构建体(pLUC3；-116至+60)的表达。对BSP启动子的进一步缺失分析表明，PGE_2可激活成纤维细胞生长因子反应元件(FRE)和cAMP反应元件(Cre)，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。FRE和垂体特异性转录因子-1调节元件(Pit-1)被确定为SMF的靶点，其作用被酪氨酸激酶抑制剂抑制。PGE_2刺激后核蛋白与放射性标记的FRE和Cre的结合增加。为了进一步表征与Cre和FRE形成的复合体中的蛋白质，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-蛋白质复合体的形成，而核提取物与抗磷酸化CREB抗体孵育则产生了可见的超位移复合体。在SMF刺激的UMR 106细胞的核提取液中，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BSP基因启动子近端的反应元件，它介导了PGE_2和SMF诱导的BSP转录。

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.