Molecular Mechanism of the differentiation of smooth muscle cells

Caldesmon （CaD）在肌动球蛋白系统中起着至关重要的作用，它分布于平滑肌细胞和非肌肉细胞中，其h（高Mr形式）-和l（低Mr形式）-CaD之间的同型相互转化是研究平滑肌细胞表型调节的有利分子事件。本研究的重点是利用CaD同型互转换作为分子标记来研究SMCs分化的分子机制。基因组分析表明，CaD亚型的表达变化受转录和独特剪接两个水平的调控；h-和l-CaDs的表达可通过外显子3内两个不同的5'剪接位点的选择性选择来调节。我们还发现，信号转导介导的alpha1beta1整合素延缓了培养的SMCs的去分化，并且alpha1beta1整合素位于分化的SMCs的细胞粘附中。在该培养体系下，h-CaD和其他分子标记物如高Mr tropom，在SMCs中维持了数天的更多的yosin和metavinculin。因此，我们利用该培养体系对SMCs中的CaD基因进行了动态分析。对原代培养的SMCs、小鼠骨骼肌细胞系（C2C12细胞）和HeLa细胞的瞬时转染实验显示，CaD启动子在SMCs中活性较高，而在其他细胞中活性极低。此外，CaD未分化的SMCs的启动子活性和蛋白质水平高于未分化的SMCs。SMCs中高水平的启动子活性依赖于一个独特的CArG盒状基序CCAAAAAAGG，位于转录起始位点上游-309至-300处，该基序及其5‘和3’侧6个非cleotide序列（CArG1）对于增强启动子活性至关重要。这些结果表明，CArG1是细胞特异性CaD基因高表达的重要顺式元件，并且CArG1的功能可能在SMCs的表型调节下受到控制。少

Caldesmon (CaD), which plays a vital role in the actomyosin system, is distributed in smooth muscle and nonmuscle cells, and its isoformal interconversion between h (high Mr form)- and l (low Mr form)-CaDs is a favorable molecular event for studying phenotypic modulation of smooth muscle cells (SMCs). This study has focused on the molecular mechanism of SMCs differentiation using such CaD isoformal interconversion as a molecular marker. Genomic analysis of CaD revealed that the expressional change in the isoforms is regulated at both levels, transcription and unique splicing ; the expression of h- and l-CaDs was regulated by alternative selection of the two distinct 5'-splice sites withn exon 3. We also found that the signal transduction mediated alpha1beta1 integrin retards the oneset of dedifferentiation of cultured SMCs and that alpha1beta1 integrin is located in cell adheadion of differentiated SMCs. Under this culture system, h-CaD and other molecular marker such as high Mr tropom … More yosin and metavinculin were maintained for several days in SMCs. Therefore, we have carried out the ptomoter anlysis of the CaD gene in SMCs using this culture system. Transient transfection assays in primary cultured SMCs, mouse skeletal muscle cell line (C2C12 cells), and HeLa cells revealed that the CaD promoter activity was high levels in SMCs, but was extremely low in other cells. In addition, the promoter activity and the protein levels of CaD indifferentiated SMCs were higher than those in dedifferentiated SMCs. High levels of the promoter activity in SMCs depended on a unique CArG box-like motif, CCAAAAAAGG,located at -309 to -300 upstream from the transcriptional starting site, and this motif in addition to its 5'-snd 3'-flanking 6 uncleotide sequences (CArG1) were essential for enhancement of the promoter activity. These results suggest that the CArG1 is an essential cis-element for cell type-specific high expression of the CaD gene and that the function of of the CArG1 might be controled under phenotypic modulation of SMCs. Less