Kim Dong-Soon, Takai Hideki, Arai Masato, Araki Shouta, Mezawa Masaru, Kawai Yoshichika, Murota Kaeko, Terao Junji, Ogata Yorimasa
Department of Periodontology, Nihon University School of Dentistry at Matsudo, Chiba 271-8587, Japan.
J Cell Biochem. 2007 Jun 1;101(3):790-800. doi: 10.1002/jcb.21233.
Quercetin is a typical flavonol-type flavonoid and is present in a variety of vegetables, and their antioxidant effect implies their possible role in the prevention of oxidative stress related chronic diseases. Bone sialoprotein (BSP) is a noncollagenous protein of the extracellular matrix in the mineralized connective tissues that has been implicated in the nucleation of hydroxyapatite crystals. Previously, we reported that isoflavone (genistein) activated BSP gene transcription is mediated through an inverted CCAAT box in the proximal BSP gene promoter. The present study investigates the regulation of BSP transcription in a rat osteoblast-like cell line, ROS 17/2.8 cells, by quercetin and its conjugated metabolite quercetin 3-glucuronide. Quercetin and quercetin 3-glucuronide (5 microM) increased the BSP mRNA levels at 12 h and quercetin upregulated the Cbfa1/Runx2 mRNA expression at 12 h. From transient transfection assays using various sized BSP promoter-luciferase constructs, quercetin increased the luciferase activity of the construct (pLUC3), including the promoter sequence nucleotides -116 to -43. Transcriptional stimulations by quercetin were almost completely abrogated in the constructs that included 2 bp mutations in the inverted CCAAT and FRE elements whereas the CCAAT-protein complex did not change after stimulation by quercetin according to gel shift assays. Quercetin increased the nuclear protein binding to the FRE and 3'-FRE. These data suggest that quercetin and quercetin 3-glucuronide increased the BSP mRNA expression, and that the inverted CCAAT and FRE elements in the promoter of the BSP gene are required for quercetin induced BSP transcription.
槲皮素是一种典型的黄酮醇类黄酮，存在于多种蔬菜中，其抗氧化作用表明它们在预防与氧化应激相关的慢性疾病中可能发挥作用。骨唾液蛋白（BSP）是矿化结缔组织细胞外基质中的一种非胶原蛋白，与羟基磷灰石晶体的成核有关。此前，我们报道异黄酮（染料木黄酮）激活BSP基因转录是通过BSP基因启动子近端的一个反向CCAAT框介导的。本研究调查了槲皮素及其共轭代谢物槲皮素3 - 葡萄糖醛酸对大鼠成骨样细胞系ROS 17/2.8细胞中BSP转录的调控。槲皮素和槲皮素3 - 葡萄糖醛酸（5 microM）在12小时时增加了BSP mRNA水平，槲皮素在12小时时上调了Cbfa1/Runx2 mRNA表达。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分析，槲皮素增加了包含启动子序列核苷酸 -116至 -43的构建体（pLUC3）的荧光素酶活性。在反向CCAAT和FRE元件中包含2 bp突变的构建体中，槲皮素的转录刺激几乎完全被消除，而根据凝胶迁移分析，槲皮素刺激后CCAAT - 蛋白质复合物没有变化。槲皮素增加了与FRE和3'-FRE的核蛋白结合。这些数据表明，槲皮素和槲皮素3 - 葡萄糖醛酸增加了BSP mRNA表达，并且BSP基因启动子中的反向CCAAT和FRE元件是槲皮素诱导BSP转录所必需的。
