| 研究実績の概要 |
We published one paper on International Journal of Molecular Sciences:
Antxr1, Which Is a Target of Runx2, Regulates Chondrocyte Proliferation and Apoptosis.
To identify Runx2 target genes in chondrocytes, we introduced Runx2-expressing adenovirus or green fluorescent protein (GFP)-expressing adenovirus into Runx2 knockout primary chondrocytes and examined the differentially expressed genes by microarray. We found that Antxr1（Tem8）was up-regulated by Runx2.
To investigate whether Tem8 expression is directly regulated by Runx2, we examined Runx2-binding regulatory regions in the Tem8 region by reanalyzing our previously published chromatin immunoprecipitation (ChIP)-seq data. In a reporter assay, Runx2 enhanced the reporter activity of 1.0-kb promoter-luciferase-0.85-kb enhancer construct. In addition, ChIP analysis using Runx2 antibody demonstrated that Runx2 binds to both Runx2-binding motifs in the 0.85-kb region. This suggested that Runx2 regulates Tem8 expression mainly through the 0.85-kb enhancer.
Further, we generated Tem8 knockout mice and chondrocyte-specific Tem8 transgenic mice. In skeletal development, the limbs of Tem8 knockout mice were shorter than wild-type mice. Tem8 transgenic mice exhibited shortened limbs. BrdU-uptake and apoptosis were both increased in chondrocytes, and the apoptosis-high regions were mineralized. These findings indicated that Tem8, of which the expression is regulated by Runx2, plays an important role in chondrocyte proliferation and that overexpression of Tem8 causes chondrocyte apoptosis accompanied by matrix mineralization.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
We already finished the first part of plans in the proposal and published the founding on a good journal.
However, the mechanism of the induction of chondrocyte apoptosis by Tem8 is not well identified. We already have appropriate mouse models, we are confident that we can study it more thoroughly.
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| 今後の研究の推進方策 |
The mechanism of the induction of chondrocyte apoptosis by Tem8 will be illuminated.
To identify Tem8 physiological ligands, we will perform pull-down assay to isolate Tem8 interacting proteins and discriminate them by mass spectrophotometry protocols. A retrovirus system will be used to overexpress a flag-tagged Tem8 fusion protein in primary chondrocytes. To capture the flag-Tem8-ligand complex, we will incubate the protein lysates with anti-flag affinity agarose beads. We will analyze the eluted complex components, first by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and later by mass spectrometry methods.
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| 次年度使用額が生じた理由 |
Commonly used in molecular biology and histological analysis such as restriction enzymes, antibody, culture media, transfection reagents, plasmid, DNA/RNA purification kit, transcription kit, reverse transcription kit, real-time PCR reagents, antibodies, gels, in situ hybridization reagents, DNA sequence reagents, pull-down kit, and MS related reagents are necessary throughout the project.
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