A novel mechanism for the regulation of osteoblast differentiation: Transcription of periostin, a member of the fasciclin I family, is regulated by the bHLH transcription factor, Twist
A novel mechanism for the regulation of osteoblast differentiation: Transcription of periostin, a member of the fasciclin I family, is regulated by the bHLH transcription factor, Twist
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DOI：
10.1002/jcb.10272
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发表时间：
2002-01-01
影响因子：
4
通讯作者：
Kudo, A
中科院分区：
文献类型：
--
作者：
Oshima, A;Tanabe, H;Kudo, A
骨膜蛋白是体外培养的早期成骨细胞以及体内骨膜和牙周韧带组织中高度表达的分泌蛋白。众所周知，在体外，骨膜蛋白支持细胞粘附和扩散。尽管人们对骨膜蛋白的转录调控机制知之甚少，但基因谱分析数据显示，Northern blot和逆转录聚合酶链反应（RT-PCR）分析证实，Twist（一种基本螺旋-环-螺旋（bHLH）转录因子）的过表达会导致骨膜蛋白表达增加。Twist是决定细胞类型和分化的重要转录因子，在早期成骨过程中发挥着重要的调节作用。小鼠颅骨原位杂交表明，骨膜蛋白和Twist mRNA在颅骨成骨前沿共定位。为了对periostin基因的5'侧翼区域进行表征，我们进行了引物延伸来确定转录起始位点，DNA序列分析证实了“Twist-box”响应元件的存在。利用MC3T3-E1细胞核提取物的电泳迁移率转移试验（EMSA）结果显示，Twist与骨膜蛋白启动子上的Twist-box序列结合。利用连接介导的PCR （LM-PCR）进行的体内足迹实验表明，Twist-box序列在未分化的MC3T3-E1成骨前细胞中受到保护，而在分化的MC3T3-E1成骨细胞中没有受到保护。为了确定Twist是否真的调控了periostin的表达，我们将periostin启动子构建体和人类Twist表达载体瞬时共转染293T细胞。报告分析表明，过表达Twist可增强骨膜蛋白启动子活性。这些数据表明，Twist可以在未分化的成骨前细胞中与骨膜蛋白启动子结合，上调骨膜蛋白的表达，这与基因谱数据中Twist上调骨膜蛋白的表达一致。(C) 2002 Wiley-Liss, Inc。
Periostin is a secreted protein that is highly expressed in early osteoblastic cells in vitro and in periosteum and periodontal ligament tissues in vivo. It is known that periostin supports cellular adhesion and spreading in vitro. Although, the mechanisms of transcriptional regulation of periostin are poorly understood, gene-profiling data have revealed that overexpression of Twist, a basic helix-loop-helix (bHLH) transcription factor, resulted in increased periostin expression as validated by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses. Twist is an important transcription factor for cell type determination and differentiation and has been shown to play an important regulatory role in early osteogenesis. In situ hybridization of mouse calvarial bones indicated that periostin and Twist mRNA are co-localized at the osteogenic fronts of calvarial bones. To characterize the 5' flanking region of the periostin gene, primer extension was carried out to identify the transcription start site, and DNA sequence analysis confirmed the presence of a 'Twist-box' response element. The results of electrophoretic mobility shift assay (EMSA) using nuclear extracts of MC3T3-E1 cells revealed that Twist bound to the Twist-box sequence on the periostin promoter. In vivo footprinting experiments using ligation-mediated PCR (LM-PCR) indicated that the Twist-box sequence was protected in undifferentiated MC3T3-E1 preosteoblasts but not in differentiated MC3T3-E1 osteoblasts. To determine whether Twist actually regulates the periostin expression, 293T cells were transiently co-transfected with the periostin promoter construct and the human Twist expression vector. Reporter analysis indicated that the periostin promoter activities were enhanced by overexpression of Twist. These data suggest that Twist can bind to the periostin promoter in undifferentiated preosteoblasts and up-regulate periostin expression, consistent with the up-regulation of periostin expression by Twist as observed in the gene-profiling data. (C) 2002 Wiley-Liss, Inc.