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

基本信息

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

项目摘要

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

骨唾液酸蛋白 (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发挥作用，甲状旁腺激素 (PTH) 通过对骨细胞和成纤维细胞生长因子 2 (FGF2) 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确定 PTH 和 FGF2 调节 BSP 的分子机制，我们分析了 PTH 或 FGF2 的作用。 ROS 17/2.8细胞中BSP表达的影响在10^-8M下，BSP mRNA的PTH刺激在3小时时首先明显(～3.8倍)，在6小时时达到最大水平(～4.7倍)。，此后缓慢下降，在 10ng/ml FGF2 时，BSP mRNA 的刺激在 3 小时（约 2.6 倍）时首次明显，并在 6 小时（约 4 小时）时达到最大水平。 PTH 和 FGF2 的作用不会改变 BSP mRNA 的稳定性。通过使用各种 BSP 启动子-荧光素酶构建体的瞬时转染测定，发现垂体特异性转录 f … More actor-1 调节元件（Pit-1； nts -111 至 -105) 和 FGF 响应元件（FRE；nts -92 至 -85）被鉴定为 PTH 和 FGF2 转录激活的靶标。在从 PTH 制备的核提取物中，抗 Pit-1 抗体识别的核蛋白对放射性标记的 Pit-1-BSP 探针的影响也有所减少。此外，ROS 细胞与双链 Pit-1 寡核苷酸的共转染也有所减少。总的来说，这些结果表明 PTH 通过涉及 cAMP 的蛋白激酶 A 途径发挥作用，通过阻断 Pit-1 相关核蛋白的作用来刺激 BSP 转录，而 Pit-1 相关核蛋白通过结合 a 蛋白来抑制 BSP 转录。因此，我们在大鼠 BSP 基因启动子中鉴定了一个新的 Pit-1 抑制元件，它是 PTH 刺激的 BSP 基因转录的靶标。ROS 17/2.8 核提取物中存在的蛋白质。细胞（但不在成纤维细胞提取物中）与包含 FRE 的双链寡核苷酸探针形成序列特异性蛋白质-DNA 复合物，并且在关键的 FGF2 刺激后增加。 FRE 序列消除了该复合物的形成并抑制了启动子活性，因此，这些研究在 BSP 基因的近端启动子中发现了一种新的 FRE，它介导组成型和 FGF2 诱导的 BSP Less 转录。