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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.

肌心蛋白（Mycd）是平滑肌细胞谱系分化所必需的，其组成型位于细胞核中，尽管其家族成员肌心蛋白相关转录因子A和B（MRTF-A/B）主要位于细胞质中并响应于Rho信号转导而易位至细胞核。他们进口核武器的机制尚不清楚。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（VSMCs）表型之间的任何相关性。在培养的VSMCs中，importin β1的敲低抑制Mycd和MRTF-A/B的核输入。体外分析表明，它们的氨基端碱性结构域（NB）是importin 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importin α/β1的亲和力也高于MRTF-A/B，并且Mycd对importin α1/β1的亲和力强于任何其他importin α/β1异二聚体。Mycd与importin α/β1的结合对G-actin不敏感，而MRTF-A/B的结合则受到G-actin不同程度的抑制。在去分化的VSMC中，importins α1和β1的水平降低，同时Mycd、血清反应因子和SMC标志物下调。相反，在分化的VSMCs中，它们的表达上调。因此，Mycd家族成员在VSMC中的核输入依赖于输入素α/β1，并且它们对输入素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标志物的表达水平相关。