Hormone and growth factor regulation of bone sialoprotein transcription and possible role of these factors on clinical application

激素和生长因子对骨唾液蛋白转录的调节及其在临床应用中的可能作用

• 批准号: 12671865
• 负责人: OGATA Yorimasa
• 金额: $ 1.47万
• 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: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671865/
• 关键词: Bone sialoprotein; Calcification; Extracellular matrix; Hormone; Growth factor; Transcriptional regulation; Tissue specificity; Gene promoter

Bone sialoprotein (BSP) is a mineralized tissue-specific protein expressed by differentiated osteoblasts that appears to function in the initial mineralization of bone. Parathyroid hormone (PTH) which regulates serum calcium through its actions on bone cells and fibroblast growth factor 2 (FGF2) is recognized as a potent mitogen for a variety of mesenchymal cells. To determine the molecular mechanism of PTH and FGF2 regulation of BSP, we analyzed the effects of the PTH or FGF2 on the expression of BSP in ROS 17/2.8 cells. At 10^<-8>M PTH stimulation of BSP mRNA was first evident at 3 h (〜3.8 -fold), reached maximal levels at 6 h (〜4.7 -fold), and declined slowly thereafter. At 10ng/ml FGF2, stimulation of BSP mRNA was first evident at 3 h (〜2.6 -fold) and reached maximal levels at 6 h (〜4 -fold).The effects of PTH and FGF2 did not alter the stability of the BSP mRNA.From transient transfection assays using various BSP promoter-luciferase constructs, a pituitary-specific transcription f … More actor-1 regulatory element (Pit-1 ; nts -111 to -105) and a FGF response element (FRE ; nts -92 to -85) were identified as a target of transcriptional activation by PTH and FGF2.Binding of a nuclear protein, recognized by anti-Pit-1 antibodies, to a radiolabelled Pit-1-BSP probe was decreased in nuclear extracts prepared from PTH. Moreover, co-transfection of ROS cells with a double-stranded Pit-1 oligonucleotide also increased luciferase activity. Collectively, these results indicate that PTH acts through a protein kinase A pathway involving cAMP to stimulate BSP transcription by blocking the action of a Pit-1-related nuclear protein that suppresses BSP transcription by binding a cognate element in the BSP promoter. Thus, we have identified a novel Pit-1 suppressor element in the rat BSP gene promoter that is the target of PTH-stimulated transcription of the BSP gene.A protein present in nuclear extracts of ROS 17/2.8 cells, but not in fibroblast extracts, formed a sequence-specific protein-DNA complex with a ds-oligonucleotide probe encompassing the FRE, and was increased following FGF2 stimulation. Several point mutations within the critical FRE sequence abrogated the formation of this complex and suppressed promoter activity. These studies, therefore, have identified a novel FRE in the proximal promoter of the BSP gene that mediates both constitutive and FGF2-induced BSP transcription. Less

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。甲状旁腺激素（PTH）通过其对骨细胞和成纤维细胞生长因子2（FGF 2）的作用来调节血清钙，被认为是多种间充质细胞的有效有丝分裂原。为了探讨PTH和FGF 2对BSP表达的调控机制，我们分析了PTH和FGF 2对ROS 17/2.8细胞中BSP表达的影响。在10 μ M时<-8>，PTH对BSP mRNA的刺激在3 h时首次明显（约3.8倍），在6 h时达到最大水平（约4.7倍），此后缓慢下降。在10 ng/ml的FGF 2浓度下，BSP mRNA的刺激作用在3 h时最明显（约2.6倍），在6 h时达到最大水平（约4.4倍）。PTH和FGF 2的作用并没有改变BSP mRNA的稳定性。从使用各种BSP启动子-荧光素酶构建体的瞬时转染实验中，发现了一种垂体特异性转录因子，它可以在细胞中表达。 ...更多信息 在PTH和FGF 2的转录激活作用中，一个作用因子-1调节元件（Pit-1 ; nts-111至-105）和一个FGF反应元件（FRE ; nts-92至-85）被鉴定为转录激活的靶点。在从PTH制备的核提取物中，被抗Pit-1抗体识别的核蛋白与放射性标记的Pit-1-BSP探针的结合减少。此外，ROS细胞与双链Pit-1寡核苷酸的共转染也增加了荧光素酶活性。总的来说，这些结果表明PTH通过涉及cAMP的蛋白激酶A途径起作用，通过阻断Pit-1相关核蛋白的作用来刺激BSP转录，所述Pit-1相关核蛋白通过结合BSP启动子中的同源元件来抑制BSP转录。因此，我们在大鼠BSP基因启动子中发现了一种新的Pit-1抑制元件，该元件是PTH刺激BSP基因转录的靶点。ROS 17/2. 8细胞核提取物中存在一种蛋白，而成纤维细胞提取物中不存在，该蛋白与包含FRE的双链寡核苷酸探针形成序列特异性蛋白-DNA复合物，并在FGF 2刺激后增加。关键的FRE序列内的几个点突变废除了这种复合物的形成并抑制了启动子活性。因此，这些研究已经在BSP基因的近端启动子中鉴定了一种新的FRE，其介导组成型和FGF 2诱导的BSP转录。少

项目成果

Emi Shimizu-Sasaki et al.: "Identification of a novel response element in the rat bone sialoprotein gene promoter that mediates constitutive and fibroblast growth factor 2-induced expression of BSP"Journal of Biologocal Chemistry. 276. 5459-5466 (2001)

Emi Shimizu-Sasaki 等人：“大鼠骨唾液酸蛋白基因启动子中介导 BSP 组成型和成纤维细胞生长因子 2 诱导表达的新型反应元件的鉴定”《生物化学杂志》。

Yorimasa Ogata et al.: "Effects of static magnetic field on osteoblasts"Japanese J Conservative Dentistry. 43. 805-811 (2000)

Yorimasa Ogata 等：“静磁场对成骨细胞的影响”日本保守牙科杂志。

Yorimasa Ogata et al.: "Parathyroid hormone regulation of bone sialoprotein gene transcription is mediated through apituitary-specific transcription factor-1 motif in the rat BSP gene promoter"Matrix Biology. 19. 395-407 (2000)

Yorimasa Ogata 等人：“骨涎蛋白基因转录的甲状旁腺激素调节是通过大鼠 BSP 基因启动子中的垂体特异性转录因子 1 基序介导的”Matrix Biology。

小方頼昌: "骨芽細胞に対する静磁場の効果"日本歯科保存学雑誌. 43. 805-811 (2000)

Yoshimasa Ogata：“静磁场对成骨细胞的影响”日本保守牙科杂志 43. 805-811 (2000)。

Emi Shimizu-Sasaki et al.: "Identification of a novel response element in the rat bone sialoprotein gene promoter that mediates constitutive and fibroblast growth factor 2-induced expression of BSP"Journal Biological Chemistry. 276. 5459-5466 (2001)

Emi Shimizu-Sasaki 等人：“大鼠骨唾液酸蛋白基因启动子中介导 BSP 组成型和成纤维细胞生长因子 2 诱导表达的新型反应元件的鉴定”《生物化学》杂志。