Smad function and intranuclear targeting share a Runx2 motif required for osteogenic lineage induction and BMP2 responsive transcription
Smad function and intranuclear targeting share a Runx2 motif required for osteogenic lineage induction and BMP2 responsive transcription
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DOI：
10.1002/jcp.20258
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发表时间：
2005-07-01
影响因子：
5.6
通讯作者：
Javed, A
中科院分区：
文献类型：
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作者：
Afzal, F;Pratap, J;Javed, A
Runx2与骨形态发生蛋白（BMP）/转化生长因子β（TGFβ）激活的Smads的协同活动对骨骼形成至关重要，但Runx2与Smad相互作用的确切结构基础尚未明确。通过缺失突变，我们确定了与BMP或TGFβ反应性Smads进行物理和功能性相互作用所需的Runx2基序。当C末端缺失至氨基酸（aa）432时，Smad反应性转录活性得以保留，但缺失至aa 391时则丧失。因此，Runx2的Smad相互作用结构域（SMID）（432 - 391）嵌入在介导核内运输的明确的核基质靶向信号（NMTS）中。当与异源Gal - 4蛋白融合时，SMID足以作为一个相互作用模块。通过共免疫沉淀研究确定，Runx2和Smad复合物的形成依赖于通过丝裂原活化蛋白激酶（MAPK）信号通路的Runx2磷酸化。我们证实所有SMID/NMTS缺失的Runx2突变体在细胞核中均未显示与Smad的原位结合，它们也不支持BMP2介导的间充质C2C12细胞系的成骨诱导。因此，我们提供了直接证据表明Runx2的SMID/NMTS结构域（391 - 432）对BMP2介导的成骨细胞分化至关重要。我们的研究结果表明，TGFβ/BMP2信号传导、MAPK依赖性磷酸化以及Runx2亚核靶向共同诱导成骨表型。
The coordinated activity of Runx2 and BMP/TGF beta-activated Smads is critical for formation of the skeleton, but the precise structural basis for the Runx2/Smad interaction has not been resolved. By deletion mutagenesis, we have defined the Runx2 motif required for physical and functional interaction with either BMP or TGF beta responsive Smads. Smad responsive transcriptional activity was retained upon deletion of the C-terminus to amino acid (aa) 432 but lost with deletion to aa 391. Thus the Smad interacting domain (SMID) of Runx2 (432-391) is embedded in the well-defined nuclear matrix targeting signal (NMTS) that mediates intranuclear trafficking. The SMID suffices as an interacting module when fused to the heterologous Gal-4 protein. Formation of the Runx2 and Smad complex is dependent on Runx2 phosphorylation through the MAPK signaling pathway, as determined by co-immunoprecipitation studies. We established that all SMID/NMTS deficient Runx2 mutants do not show in situ association with Smad in the nucleus nor do they support BMP2-mediated osteogenic induction of the mesenchymal C2C12 cell line. Thus, we provide direct evidence that the SMID/NMTS domain (391-432) of Runx2 is essential for BMP2-mediated osteoblast differentiation. Our findings suggest that TGF beta/ BMP2 signaling, MAPK dependent phosphorylation, and Runx2 subnuclear targeting converge to induce the osteogenic phenotype.