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

骨涎蛋白（BSP）是一种矿化组织特异性蛋白，由分化成骨细胞表达，似乎在骨的初始矿化中起作用。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）的作用调节血清钙，被认为是多种间充质细胞的有效丝裂原。为了确定PTH和FGF2调控BSP的分子机制，我们分析了PTH或FGF2对ROS 17/2.8细胞中BSP表达的影响。在10^<-8>M时，PTH对BSP mRNA的刺激在3 h时首先明显（~ 3.8倍），在6 h时达到最大水平（~ 4.7倍），此后缓慢下降。当FGF2浓度为10ng/ml时，BSP mRNA的刺激在3 h时首先明显（约2.6倍），在6 h时达到最大水平（约4倍）。PTH和FGF2的作用没有改变BSP mRNA的稳定性。通过使用多种BSP启动子-荧光素酶构建体进行瞬时转染实验，我们确定了PTH和FGF2转录激活的靶标是垂体特异性转录的More actor-1调控元件（Pit-1； nts -111至-105）和FGF应答元件（FRE； nts -92至-85）。PTH提取的核提取物降低了抗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。此外，用双链Pit-1寡核苷酸共转染ROS细胞也增加了荧光素酶的活性。总的来说，这些结果表明PTH通过一个涉及cAMP的蛋白激酶a途径，通过阻断pit -1相关核蛋白的作用来刺激BSP转录，该核蛋白通过结合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个新的Pit-1抑制元件，该元件是pth刺激的BSP基因转录的靶点。在ROS 17/2.8细胞的核提取物中存在一种蛋白，但在成纤维细胞提取物中不存在，它与包含FRE的ds-寡核苷酸探针形成序列特异性蛋白- dna复合物，并在FGF2刺激后增加。关键FRE序列中的几个点突变取消了该复合体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发现了一个新的FRE，该FRE介导构成型和fgf2诱导的BSP转录。少

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