Shimizu Emi, Ogata Yorimasa
Department of Endodontics, Research Institute of Oral Science, Nihon University School of Dentistry at Matsudo, Chiba, 271-8587, Japan.
J Cell Biochem. 2002;86(1):35-44. doi: 10.1002/jcb.10194.
Bone sialoprotein (BSP) is a major noncollagenous protein of the mineralized bone extracellular matrix that has been implicated in the nucleation of hydroxyapatite. Recent studies have shown that BSP is also expressed by osteotropic cancers suggesting BSP might play a role in the pathogenesis of bone metastases. The present study investigates regulation of BSP transcription in rat osteosarcoma ROS 17/2.8 cells by flavonoids: genistein (an inhibitor of protein tyrosine kinases), daidzein (an inactive compound of genistein), flavone, and flavanone. Genistein, daidzein, and flavone (50 microM) increased steady state levels of BSP mRNA about 1.7-fold at 12 h. From transient transfection assays using various sized BSP promoter-luciferase constructs, genistein increased luciferase activities within 12 h. Constructs including the promoter sequence nucleotides (nts) -116 to -43 (pLUC3) were found to enhance transcriptional activity approximately 2.6-fold in ROS 17/2.8 cells treated with genistein (50 microM). Daidzein, flavone, and flavanone (50 microM) also increased luciferase activities. In contrast, the tyrosine kinase inhibitors, herbimycin A and lavendustin A, which do not have a flavonoid structure, did not stimulate BSP transcription. Transcriptional stimulation by genistein was almost completely abrogated in a construct that included 2 bp mutations in the inverted CCAAT box. A monoclonal antibody against NF-YA, a CCAAT box-binding transcription factor, inhibited formation of DNA-NF-Y protein complex in gel shift assays formed by nuclear extracts of ROS 17/2.8 cells. These data suggest that the inverted CCAAT box is required for flavonoid-induced BSP expression and that the stimulatory action is dependent on the flavone structure and does not involve an inhibitory action on protein tyrosine kinase.
骨唾液蛋白（BSP）是矿化骨细胞外基质中的一种主要非胶原蛋白，与羟基磷灰石的成核作用有关。最近的研究表明，促骨癌细胞也表达BSP，这表明BSP可能在骨转移的发病机制中起作用。本研究调查了黄酮类化合物（染料木黄酮，一种蛋白酪氨酸激酶抑制剂；大豆苷元，染料木黄酮的无活性化合物；黄酮和黄烷酮）对大鼠骨肉瘤ROS 17/2.8细胞中BSP转录的调节作用。染料木黄酮、大豆苷元和黄酮（50微摩尔）在12小时时使BSP mRNA的稳态水平增加了约1.7倍。通过使用各种大小的BSP启动子-荧光素酶构建体进行瞬时转染实验，染料木黄酮在12小时内增加了荧光素酶活性。发现在用染料木黄酮（50微摩尔）处理的ROS 17/2.8细胞中，包括启动子序列核苷酸（nts）-116至-43（pLUC3）的构建体可使转录活性增强约2.6倍。大豆苷元、黄酮和黄烷酮（50微摩尔）也增加了荧光素酶活性。相比之下，不具有黄酮类结构的酪氨酸激酶抑制剂赫曲霉素A和拉文达ustin A并未刺激BSP转录。在一个包含反向CCAAT框中2个碱基对突变的构建体中，染料木黄酮的转录刺激作用几乎完全被消除。一种针对CCAAT框结合转录因子NF-YA的单克隆抗体在凝胶迁移实验中抑制了由ROS 17/2.8细胞核提取物形成的DNA-NF-Y蛋白复合物的形成。这些数据表明，反向CCAAT框是黄酮类化合物诱导BSP表达所必需的，且刺激作用依赖于黄酮结构，并不涉及对蛋白酪氨酸激酶的抑制作用。
