Seth A, Lee B K, Qi S, Vary C P
MRC Group in Periodontal Physiology, and the Laboratory of Medicine and Pathobiology, University of Toronto, Sunnybrook, Canada.
J Bone Miner Res. 2000 Sep;15(9):1683-96. doi: 10.1359/jbmr.2000.15.9.1683.
To achieve new insights into the coordinate regulation of gene expression during osteoblast differentiation we utilized an approach involving global analysis of gene expression to obtain the identities of messenger RNAs (mRNAs) expressed using an established in vitro model of bone development. MC3T3-E1 osteoblast-like cells were induced to differentiate by the addition of beta-glycerophosphate (beta-GP) and ascorbic acid. RNA samples derived from induced and uninduced control MC3T3-E1 cells were used to prepare complementary DNA (cDNA) for serial analysis of gene expression (SAGE). A preliminary SAGE database was produced and used to prepare a hybridization array to further facilitate the characterization of changes in the expression levels of 92 of the SAGE-mRNA assignments after induction of osteoblast differentiation, specifically after 6 days and 14 days of ascorbate treatment. SAGE-array hybridization analysis revealed coordinate induction of a number of mRNAs including Rab24, calponin, and calcyclin. Levels of MSY-1, SH3P2, fibronectin, alpha-collagen, procollagen, and LAMPI mRNAs, present at day 6 postinduction, were markedly reduced by day 14 postinduction. A number of unanticipated and potentially important developmental genes were identified including the transforming growth factor beta (TGF-beta) superfamily member Lefty-1. Lefty-1 transcript and translation product were found to be induced during the course of MC3T3-E1 cell differentiation. We present evidence, using transient transfection and antibody neutralization approaches, that Lefty-1 modulates the induction of alkaline phosphatase (ALP) after treatment of MC3T3-E1 cells with ascorbate and beta-GP. These data should provide useful new information for future analysis of transcriptional events in osteoblast differentiation and mineralization.
为了深入了解成骨细胞分化过程中基因表达的协同调控，我们采用了一种方法，该方法涉及对基因表达进行全局分析，以确定在已建立的骨发育体外模型中表达的信使核糖核酸（mRNA）的身份。通过添加β-甘油磷酸酯（β-GP）和抗坏血酸诱导MC3T3-E1成骨样细胞分化。从诱导和未诱导的对照MC3T3-E1细胞中提取的RNA样本用于制备互补DNA（cDNA），以进行基因表达的序列分析（SAGE）。构建了一个初步的SAGE数据库，并用于制备杂交阵列，以进一步促进对成骨细胞分化诱导后，特别是抗坏血酸处理6天和14天后，92个SAGE-mRNA赋值的表达水平变化的表征。SAGE阵列杂交分析揭示了包括Rab24、钙调蛋白和钙周期蛋白在内的一些mRNA的协同诱导。诱导后第6天存在的MSY-1、SH3P2、纤连蛋白、α-胶原蛋白、前胶原蛋白和LAMPI mRNA水平在诱导后第14天显著降低。鉴定出了一些意想不到的且可能重要的发育基因，包括转化生长因子β（TGF-β）超家族成员Lefty-1。发现Lefty-1转录本和翻译产物在MC3T3-E1细胞分化过程中被诱导。我们使用瞬时转染和抗体中和方法提供证据表明，Lefty-1在用抗坏血酸和β-GP处理MC3T3-E1细胞后调节碱性磷酸酶（ALP）的诱导。这些数据应为未来成骨细胞分化和矿化过程中转录事件分析提供有用的新信息。
