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STUDY OF BONE FORMATION SIGNALING IN WNT-LRP-DKK AND PROTEOSOME SYSTEM

WNT-LRP-DKK 和蛋白质体系统中骨形成信号传导的研究

基本信息

批准号：
16390529
负责人：
TAMURA Masato
金额：
$ 8.7万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16390529/
关键词：
osteoblast Wnt BMP-2 LRP Dkk 骨形成 OPG BMP LRP5 シグナル伝達

项目摘要

Wnt/β-catenin signaling plays a role in the developing skeletal system. However, the exact mechanisms by which Wnt/β-catenin signaling regulates bone remodeling remain to be elucidated. In this study, we demonstrated that an interaction between β-catenin and Smad was identified and found to be crucial for Wnt-mediated regulation of the BMP-2 responsive gene expression, and BMP-2 up-regulated Wnt-induced lymphoid enhancing factor 1/T cell factor (Lef1/Tcf)-dependent TopFlash transcriptional activity in C2C12 cell line. Also, Wnt/β-catenin signaling regulates expression of osteoprotegerin (OPG) and receptor activator of NFkB ligand (RANKL) in osteoblasts. Moreover, we investigated the mechanism of how induces OPG expression by Wnt/β-catenin signaling in osteoblasts. OPG expression was induced by over-expression of Wnt3a or activated β-catenin in mesenchymal pluripotent C2C12 cells or osteoblastic MC3T3-E1 cells. In these cultures, BMP-2 synergistically enhanced their OPG expression. Sile … More ncing of glycogen synthase kinase-3β by siRNA or sgRNA (tRNaseZL-utilizing gene silencing method) also increased OPG levels of culture supernatant in C2C12 cells estimated by ELISA assay. To investigate the mechanisms of the Wnt/β-catenin signaling activated OPG gene transcription, we have cloned approximately 1.5-kilobase pair genomic DNA fragment corresponding to the 5'-flanking promoter region of the murine OPG gene. Activated β-catenin results in 10-15 fold increase in reporter gene transcription activity of the 1.5-kilobase fragment by transfection assay performed in C2C12 cells. Deletion analyses revealed that a proximal 253-base pair region in the promoter was required for Wnt/β-catenin responsiveness. In this region, we identified four putative Lef1/Tcf binding sites, which meet with a consensus sequence, reported to be recognized by Lef1/Tcf proteins. Using site-directed mutation analyses, functional Lef1/Tcf binding sites was identified in their region. Smad protein involved and interacted with their Wnt/β-catenin responsive element on the OPG promoter in response to BMP-2 stimulation. Chromatin IP assay indicated in viro evidence that β-catenin regulates transcription of OPG via promoter region of their site. These results show that OPG is a target gene for Wnt/β-catenin signaling and their induction is mediated novel Wnt/β-catenin response element of the OPG gene promoter. Less

Wnt/β-catenin信号在骨骼系统发育中发挥作用。然而，Wnt/β-catenin信号调节骨重塑的确切机制仍有待阐明。在这项研究中，我们发现β-catenin和Smad之间的相互作用对wnt介导的BMP-2应答基因表达的调控至关重要，BMP-2上调wnt诱导的淋巴细胞增强因子1/T细胞因子（Lef1/Tcf）依赖性TopFlash在C2C12细胞系中的转录活性。此外，Wnt/β-catenin信号传导调节成骨细胞中骨保护素（OPG）和NFkB配体受体激活剂（RANKL）的表达。此外，我们还研究了Wnt/β-catenin信号在成骨细胞中诱导OPG表达的机制。在间充质多能性C2C12细胞或成骨细胞MC3T3-E1细胞中，通过过表达Wnt3a或活化β-catenin诱导OPG的表达。在这些培养中，BMP-2协同增强了它们的OPG表达。通过siRNA或sgRNA （trnasezl利用基因沉默方法）增强糖原合成酶激酶3β，也可以通过ELISA检测提高C2C12细胞培养上清的OPG水平。为了研究Wnt/β-catenin信号通路激活OPG基因转录的机制，我们克隆了大约1.5千碱基对的基因组DNA片段，对应于小鼠OPG基因5'侧启动子区域。通过在C2C12细胞中进行转染实验，激活β-catenin导致1.5千碱基片段的报告基因转录活性增加10-15倍。缺失分析显示，启动子中近253个碱基对区域是Wnt/β-catenin响应所必需的。在这个区域，我们确定了四个假定的Lef1/Tcf结合位点，它们符合公认的序列，被报道为Lef1/Tcf蛋白识别。利用位点定向突变分析，在其所在区域确定了功能性的Lef1/Tcf结合位点。Smad蛋白参与并与OPG启动子上的Wnt/β-catenin响应元件相互作用，以响应BMP-2刺激。染色质IP实验表明，β-catenin通过OPG位点的启动子区调控OPG的转录。这些结果表明，OPG是Wnt/β-catenin信号转导的靶基因，其诱导是通过OPG基因启动子的新型Wnt/β-catenin应答元件介导的。少