2006 Fiscal Year Final Research Report Summary
Transcriptional regulation of osteogenesis using siRNA and its application to bone formation
| Project/Area Number |
17300153
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biomedical engineering/Biological material science
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| Research Institution | Osaka University |
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Principal Investigator |
KANEDA Yasufumi Osaka University, Graduate School of Medicine, Professor, 医学系研究科, 教授 (10177537)
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| Project Period (FY) |
2005 – 2006
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| Keywords | BMP / Twist-1 / siRNA / Idl / Smad / E47 / osteogenesis / histone deacetylase |
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| Research Abstract |
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
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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
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Research Products
(21 results)
