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

• 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

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 (BMP) 通过激活 Smad 转定位到细胞核中，与多种转录调节蛋白结合并控制基因表达。也称为与 Smads 相互作用的关键分子。在此，我们发现间充质分化的负调节因子 Twist-1 的过度表达可抑制 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 Twist-1 在受体激活后直接与 Smads 相互作用的活性，并抑制了转录。为了最大限度地抑制 BMP 信号传导，Twist-1 需要与 E47 形成异二聚体，从而大大延长了 BMP 信号的半衰期。 Twist-1。此外，Idl 通过诱导 Twist-1 降解克服了 Twist-1 对 BMP 信号传导的抑制作用。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。和 Idl 可以通过抑制 Twist-1 功能的正反馈回路来调节 BMP 信号传导，因此这两种分子可能通过控制来调节间充质细胞分化为后代，例如成骨细胞。 BMP/Smad 信号传导 为了分析 Twist-1 抑制 BMP 信号传导的机制，进行了免疫共沉淀研究，结果显示 Twist-1 与 MC3T3 中的 Smad4 和组蛋白脱乙酰酶 (HDAC) 1 形成复合物。 -E1细胞稳定表达Twist-1，含有HDAC抑制剂曲古抑菌素、碱性磷酸酶等成骨因子，这些结果表明，Twist-1 通过将 HDAC1 招募到 Smad4 来抑制 BMP 信号传导，表明 Twist-1 对 Smad 与靶 DNA 序列的结合没有影响。

项目成果

E2F decoy oligodeoxynucleotide ameliorates cartilageinvasion by infiltrating synovium derived rheumatoid arthritis.

E2F 诱饵寡脱氧核苷酸通过浸润滑膜衍生的类风湿性关节炎改善软骨侵袭。

• 发表时间: 2006
• 期刊: Int. J. Mol.Med. 18・2
• 作者: Tomita;T.;et al.;Kaneda;Y.;Yoshikawa;H.
• 通讯作者: H.

Gene Transfer, a laboratory manual

基因转移，实验室手册

• 发表时间: 2007
• 作者: Hayashi;M. et al.;Yasufumi Kaneda
• 通讯作者: Yasufumi Kaneda

Dnmt3a2 targets endogenous Dnmt3L to ES cell chromatin and induces regional DNA methylation

• DOI: 10.1111/j.1365-2443.2006.01012.x
• 发表时间: 2006-10-01
• 期刊: GENES TO CELLS
• 影响因子: 2.1
• 作者: Nimura, Keisuke;Ishida, Chisaki;Kaneda, Yasufumi
• 通讯作者: Kaneda, Yasufumi

Non-viral Vectors for Gene Therapy

• DOI: 10.1007/978-3-030-41333-0_2
• 发表时间: 2020
• 期刊: A Handbook of Gene and Cell Therapy
• 作者: Clévio Nóbrega;Liliana S. Mendonça;Carlos A. Matos

Biocompatible polymer enhances the in vitro and in vivo transfection efficiency of HVJ envelope vector

• DOI: 10.1002/jgm.735
• 发表时间: 2005-07
• 期刊: The Journal of Gene Medicine
• 作者: Hidetoshi Mima;Ryuji Tomoshige;Toshihide Kanamori;Y. Tabata;Seiji Yamamoto;S. Ito;K. Tamai;Y. Kaneda