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

肌钙蛋白(myocardin，Mycd)是肌钙蛋白(Myocardin，Mycd)家族成员，在Rho信号作用下，肌钙蛋白A和肌钙蛋白相关转录因子B(myocardin-Related转录因子A/B，MRTF-A/B)主要存在于胞浆中，并转位到胞核，Mycd是平滑肌细胞分化所必需的。他们的核进口机制尚不清楚。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)的表型之间的任何相关性。在培养的VSMC中，Importinβ1的敲除抑制了MYCD和MRTFA/B的核输入。体外分析表明，它们的NH2末端碱性结构域(NB)是Importinα/β1的结合部位。然而，即使在没有G-肌动蛋白的情况下，MyCD对Importinα/β1的亲和力也高于mRTF-A/B，并且MyCD对Importinα1/β1的亲和力强于任何其他Importinα/β1异源二聚体。MYCD与Importinα/β-1的结合对G-肌动蛋白不敏感，而MRTFA/B对G-肌动蛋白的抑制作用不同。在去分化的VSMC中，随着MYCD、血清反应因子和SMC标志物的下调，α-1和β-1的表达水平降低。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Importinα/β1，而它们与Importinα/β1异源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表型依赖的SMC标志物的表达水平有关。

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.