Shimizu-Sasaki Emi, Yamazaki Muneyoshi, Furuyama Shunsuke, Sugiya Hiroshi, Sodek Jaro, Ogata Yorimasa
Department of Endodontics, Nihon University School of Dentistry at Matsudo, Chiba, Japan.
Connect Tissue Res. 2003;44 Suppl 1:103-8.
Bone sialoprotein (BSP), an early marker of osteoblast differentiation, has been implicated in the nucleation of hydroxyapatite during de novo bone formation. Basic fibroblast growth factor (FGF2) is recognized as a potent mitogen for a variety of mesenchymal cells. In skeletal tissues, FGF2 produced by osteoblasts accumulates in the bone matrix and acts as an autocrine/paracrine regulator of bone cells. To determine the molecular mechanism of FGF2 regulation of osteogenesis, we have analyzed the effects of FGF2 on the expression of BSP in the rat osteosarcoma cell line ROS 17/2.8. FGF2 at 10 ng/ml, increased BSP mRNA levels approximately 4-fold; the stimulation was first evident at 3 hr, reached maximal levels at 6 hr. The stability of the BSP mRNA was not significantly affected by FGF2, suggesting that the increased mRNA was due to increased transcription. From transient transfection analyses using various BSP promoter-luciferase constructs, a FGF2 response element (FRE) (nts -92 to -85, "GGTGAGAA") was identified as a target of transcriptional activation by FGF2. Ligation of two copies of the FRE 5' to an SV40 promoter was sufficient to confer FGF responsive transcription. A sequence-specific protein-DNA complex, formed with a double-stranded oligonucleotide encompassing the FRE and nuclear extracts from ROS 17/2.8 cells, but not from fibroblasts, was increased following FGF2 stimulation. Several point mutations within the critical FRE sequence abrogated the formation of this complex and suppressed both basal and FGF2-mediated promoter activity. Thus, we have identified a novel FRE within the proximal promoter of the BSP gene that mediates both constitutive and FGF2-induced BSP transcription.
骨唾液蛋白（BSP）是成骨细胞分化的早期标志物，在新生骨形成过程中与羟基磷灰石的成核有关。碱性成纤维细胞生长因子（FGF2）被认为是多种间充质细胞的有效促有丝分裂原。在骨骼组织中，成骨细胞产生的FGF2积聚在骨基质中，并作为骨细胞的自分泌/旁分泌调节因子。为了确定FGF2调节成骨作用的分子机制，我们分析了FGF2对大鼠骨肉瘤细胞系ROS 17/2.8中BSP表达的影响。10 ng/ml的FGF2使BSP mRNA水平增加约4倍；这种刺激在3小时时首次明显，在6小时时达到最高水平。FGF2对BSP mRNA的稳定性没有显著影响，这表明mRNA的增加是由于转录增加。通过使用各种BSP启动子-荧光素酶构建体的瞬时转染分析，FGF2反应元件（FRE）（核苷酸-92至-85，“GGTGAGAA”）被确定为FGF2转录激活的靶点。将两个FRE拷贝连接到SV40启动子的5'端足以赋予FGF反应性转录。用包含FRE的双链寡核苷酸与ROS 17/2.8细胞而非成纤维细胞的核提取物形成的序列特异性蛋白质-DNA复合物，在FGF2刺激后增加。关键FRE序列内的几个点突变消除了这种复合物的形成，并抑制了基础和FGF2介导的启动子活性。因此，我们在BSP基因近端启动子内鉴定了一个新的FRE，它介导组成型和FGF2诱导的BSP转录。
