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Transcriptional regulation of osteogenesis using siRNA and its application to bone formation

siRNA对成骨的转录调控及其在骨形成中的应用

基本信息

批准号：
17300153
负责人：
KANEDA Yasufumi
金额：
$ 7.55万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17300153/
关键词：
BMP Twist-1 siRNA Idl Smad E47 osteogenesis histone deacetylase Id1 骨分化 HVJ envelope vector

项目摘要

Bone morphogenetic proteins (BMPs) transduce signals through the activation of Smads. Activated Smads translocate into the nucleus, where they associate with a diverse group of transcriptional regulatory proteins and control gene expression. The basic helix-loop-helix (bHLH) transcription factors are also known as key molecules interacting with Smads. Herein we showed that overexpression of Twist-1, a negative regulator of mesenchymal differentiation, suppressed BMP-induced osteoblast differentiation. Then, we constructed Twist-1 specific short interfering RNA (siRNA) to downregulate Twist-1. Inhibition of endogenous Twist-1 by the siRNA enhanced the activity of BMP signaling. Twist-1 interacted directly with Smads after receptor activation-and inhibited transcriptional activity mediated by Smads. For maximal inhibition of BMP signaling, Twist-1 required heterodimerization with E47, resulting in a greatly extended half-life for Twist-1. Furthermore, the inhibitory effect of Twist-1 on … More BMP signaling was overcome by Idl through the induction of Twist-1 degradation. These findings suggest that Twist-1 can act as an inhibitor of BMP signaling through interaction with Smads, and Idl can regulate BMP signaling through a positive feedback loop repressing Twist-1 function. These two molecules may therefore regulate differentiation of mesenchymal cells into progeny such as osteoblasts by controlling BMP/Smad signaling. To analyze the mechanism of the inhibition of BMP signaling by Twist-1, co-immunoprecipitation study was conducted. Co-immunoprecipitation assay revealed that Twist-1 formed a complex with Smad4 and histone deacetylase (HDAC) 1 in MC3T3-E1 cells stably expressing Twist-1. With trichostatin, an HDAC inhibitor, osteogenic factors such as alkaline phosphatase, Runx2 and osteopontin increased. Those results suggested that Twist-1 inhibited BMP signaling by recruiting HDAC1 to Smad4. Gel-shift assay showed that Twist-1 had no effect on the binding of Smads to the target DNA sequence. Less

骨形态发生蛋白（BMPs）通过激活Smads来传递信号。激活的Smads易位到细胞核中，在那里它们与一组不同的转录调节蛋白结合并控制基因表达。碱性螺旋-环-螺旋（bHLH）转录因子也被称为与Smads相互作用的关键分子。在此，我们发现Twist-1的过度表达，一种间充质分化的负调节因子，抑制BMP诱导的成骨细胞分化。然后，我们构建了Twist-1特异性短干扰RNA（siRNA）来下调Twist-1。siRNA对内源性Twist-1的抑制增强了BMP信号传导的活性。Twist-1在受体激活后直接与Smads相互作用，并抑制Smads介导的转录活性。为了最大限度地抑制BMP信号传导，Twist-1需要与E47异二聚化，导致Twist-1的半衰期大大延长。此外，Twist-1对 ...更多信息 BMP信号传导被Idl通过诱导Twist-1降解克服。这些发现表明Twist-1可以通过与Smads相互作用作为BMP信号传导的抑制剂，并且Idl可以通过抑制Twist-1功能的正反馈环来调节BMP信号传导。因此，这两种分子可以通过控制BMP/Smad信号传导来调节间充质细胞向后代如成骨细胞的分化。为了分析Twist-1抑制BMP信号传导的机制，进行免疫共沉淀研究。免疫共沉淀实验表明，在稳定表达Twist-1的MC 3 T3-E1细胞中，Twist-1与Smad 4和组蛋白去乙酰化酶（HDAC）1形成复合物。使用HDAC抑制剂阿司他汀，成骨因子如碱性磷酸酶、Runx 2和骨桥蛋白增加。这些结果表明，Twist-1通过将HDAC 1募集到Smad 4来抑制BMP信号传导。凝胶迁移实验表明Twist-1对Smads与靶DNA序列的结合没有影响。少