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

调控成骨细胞分化的分子机制一直是骨生物学研究的重要课题之一。细胞的分化在转录水平上受到各种类型的转录因子的控制，这些转录因子已经通过生化和遗传手段鉴定出来，并且在翻译水平上受到控制。之前，我报道了螺旋-环-螺旋型蛋白参与成骨细胞表型表达，如骨钙素，并因此分化。在本研究中，我研究了骨钙素基因表达和RNA结合蛋白在成骨细胞分化中的翻译调控。通过蔗糖梯度离心的多体分析，我发现结合骨钙素mRNA的核糖体在成骨细胞分化过程中被上调。然后，我克隆了小鼠骨钙素基因，构建了包括骨钙素基因5′或3′非翻译区（UTR）在内的各种报告质粒。我将这些报告质粒转染到间充质未分化细胞中，并加入骨形态发生蛋白，然后测量荧光素酶的活性。这些实验表明骨钙素基因UTR的几个区域参与了成骨细胞骨钙素基因的翻译调控。我还通过凝胶转移分析证明了RNA结合蛋白与骨钙素基因UTR的几个区域结合。这些结果表明，一些与骨钙素基因UTR相互作用的RNA结合蛋白在成骨细胞分化中起重要作用。

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.