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

骨形态发生蛋白（BMPs）通过Smads的激活来传递信号。激活的Smads转运到细胞核中，在那里它们与多种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。本研究表明，Twist-1（间充质分化负调节因子）的过表达抑制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抑制内源性Twist-1可增强BMP信号的活性。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，Twist-1需要与E47异源二聚化，导致Twist-1的半衰期大大延长。此外，Idl通过诱导Twist-1降解，克服了Twist-1对BMP信号的抑制作用。这些发现表明，Twist-1可以通过与Smads的相互作用作为BMP信号传导的抑制剂，而Idl可以通过抑制Twist-1功能的正反馈回路调节BMP信号传导。因此，这两种分子可能通过控制BMP/Smad信号来调节间充质细胞向子代细胞（如成骨细胞）的分化。为了分析Twist-1抑制BMP信号传导的机制，我们进行了免疫共沉淀研究。共免疫沉淀实验显示，Twist-1在稳定表达Twist-1的MC3T3-E1细胞中与Smad4和组蛋白去乙酰化酶（HDAC） 1形成复合物。使用HDAC抑制剂曲古斯汀后，成骨因子如碱性磷酸酶、Runx2和骨桥蛋白增加。这些结果表明Twist-1通过将HDAC1募集到Smad4来抑制BMP信号传导。凝胶转移实验表明Twist-1对Smads与目标DNA序列的结合没有影响。少

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