BINDING PROTEINS FOR UTR OF OSTEOCALCIN GENE AND THESE FUNCTIONS IN OSTEOBLASTC DIFFERENTIATION

骨钙素基因UTR结合蛋白及其在成骨细胞分化中的功能

• 批准号: 11671847
• 负责人: TAMURA Masato
• 金额: $ 2.3万
• 依托单位: KAGOSHIMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671847/
• 关键词: OSTEOBLAST; OSTEOCALCIN; GENE EXPRESSION; OSTEOBLAST; OSTEOCALCIN; GENE EXPRESSION

Elucidation of molecular mechanisms controlling differentiation of osteoblasts has been one of the major subjects in bone biology. Differentiation of cells is controlled at the level of transcription by various classes of transcription factors that have been identified through biochemical and genetic means, and the level of translation. Previously, I reported that helix-loop-helix type of proteins are involved in osteoblastic phenotype expression, such as osteocalcin, and hence differentiation. In this study, I investigated the translational regulation of osteocalcin gene expression and RNA binding proteins in osteoblastic differentiation. By polysome analysis using sucrose gradient centrifugion, I found that ribosomes that bind osteocalcin mRNA are up-regulated by osteoblastic differentiation. Then, I cloned mouse osteocalcin gene and made various constructions of reporter plasmids including the 5' or 3' of untranslated region (UTR) of osteocalcin gene. I transfected these reporter plasmids to mesenchymal undifferentiated cells and added bone morphogenetic protein, then measured luciferase activities. These experiments reveals that several regions of UTR of osteocalcin gene are involved the translational regulation of osteocalcin gene in osteoblasts. I also demonstrated that RNA binding proteins binding to the several regions of UTR of osteocalcin gene by gel shift analyses. These results suggested that some RNA binding proteins that interact UTR of osteocalcin gene play important roles for osteoblastic differentiation.

阐明控制成骨细胞分化的分子机制一直是骨生物学的主要学科之一。通过生化和遗传手段鉴定出的各种转录因子以及翻译水平，通过各种转录因子来控制细胞的分化。以前，我报道了螺旋 - 环螺旋类型的蛋白质参与成骨细胞表达的表达，例如骨钙素，因此分化。在这项研究中，我研究了成骨细胞分化中骨钙蛋白基因表达和RNA结合蛋白的翻译调节。通过使用蔗糖梯度离心分析，我发现结合骨钙素mRNA的核糖体被成骨细胞分化上调。然后，我克隆小鼠骨钙素基因，并制造了记者质粒的各种结构，包括骨钙素基因的未翻译区（UTR）的5'或3'。我将这些报告基因质粒转染到间充质未分化的细胞中，并添加了骨形态发生蛋白，然后测量了荧光素酶活性。这些实验表明，骨钙素基因的UTR几个区域涉及成骨细胞中骨钙素基因的转化调节。我还证明了通过凝胶移位分析与骨钙蛋白基因UTR的多个区域结合的RNA结合蛋白。这些结果表明，与骨钙素基因相互作用的一些RNA结合蛋白在成骨细胞分化方面起着重要作用。