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Regulatory mechanism for the function of myocardin family members and its relation to cell phenotype

心肌素家族成员功能的调控机制及其与细胞表型的关系

基本信息

批准号：
20590279
负责人：
HAYASHI Ken'ichiro
金额：
$ 3万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2008
资助国家：
日本
起止时间：
2008 至 2010
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-20590279/
关键词：
myocardin MRTF-A B importin α β1 G-actin 核移行平滑筋細胞形質転換 MRTF-A/B importin α/β1 basic domain myocardin family members importinα importin 核移行シグナル

项目摘要

Myocardin (Mycd), which is essential for the differentiation of the smooth-muscle cell lineage, is constitutively located in the nucleus, although its family members, myocardin-related transcription factors A and B (MRTF-A/B), mostly reside in the cytoplasm and translocate to the nucleus in response to Rho-signaling. The mechanism for their nuclear import is unclear. Here, we investigated the mechanism for the nuclear import of Mycd family members, and demonstrated any correlation between such mechanism and the phenotype of vascular smooth muscle cells (VSMCs). In cultured VSMCs, the knockdown of importin β1 inhibited the nuclear import of Mycd and MRTF-A/B. Their NH2-terminal basic domain (NB) was identified as a binding site for importin α/β1 by in vitro analyses. However, Mycd had a higher affinity for importin α/β1 than MRTF-A/B did, even in the absence of G-actin, and Mycd's affinity for importin α1/β1 was stronger than for any other importin α/β1 heterodimers. The binding of Mycd to importin α/β1 was insensitive to G-actin, whereas that of MRTF-A/B was differently inhibited by G-actin. In dedifferentiated VSMCs, the levels of importins α1 and β1 were reduced concomitant with down-regulation of Mycd, serum response factor, and SMC markers. By contrast, in differentiated VSMCs, their expressions were up-regulated. Thus, the nuclear import of Mycd family members in VSMCs depends on importin α/β1, and their relative affinities for importin α/β1 heterodimers determine the Mycds' nuclear import. The expression of the Mycds' nuclear import machineries is related to the expression levels of VSMC phenotype-dependent SMC markers.

心肌素 (Mycd) 对平滑肌细胞谱系的分化至关重要，其组成型位于细胞核中，尽管其家族成员心肌素相关转录因子 A 和 B (MRTF-A/B) 主要存在于细胞质中，并响应 Rho 信号转位至细胞核。它们的核输入机制尚不清楚。在这里，我们研究了 Mycd 家族成员的核输入机制，并证明了这种机制与血管平滑肌细胞 (VSMC) 表型之间的相关性。在培养的 VSMC 中，输入蛋白 β1 的敲低抑制了 Mycd 和 MRTF-A/B 的核输入。通过体外分析，它们的 NH2 末端基本结构域 (NB) 被鉴定为输入蛋白 α/β1 的结合位点。然而，即使在没有 G-肌动蛋白的情况下，Mycd 对输入蛋白 α/β1 的亲和力也比 MRTF-A/B 更高，并且 Mycd 对输入蛋白 α1/β1 的亲和力比任何其他输入蛋白 α/β1 异二聚体更强。 Mycd 与输入蛋白 α/β1 的结合对 G-肌动蛋白不敏感，而 MRTF-A/B 的结合则受到 G-肌动蛋白不同程度的抑制。在去分化的 VSMC 中，输入蛋白 α1 和 β1 的水平随着 Mycd、血清反应因子和 SMC 标志物的下调而降低。相比之下，在分化的 VSMC 中，它们的表达上调。因此，VSMC 中 Mycd 家族成员的核输入取决于输入蛋白 α/β1，并且它们与输入蛋白 α/β1 异二聚体的相对亲和力决定了 Mycd 的核输入。 Mycds 核输入机制的表达与 VSMC 表型依赖性 SMC 标记的表达水平相关。