Molecular Mechanism of the differentiation of smooth muscle cells

钙调素（Caldesmon，CaD）在肌动球蛋白系统中起重要作用，分布于平滑肌和非肌细胞中，其在h（高分子量形式）-CaD和l（低分子量形式）-CaD之间的异构转化是研究平滑肌细胞（SMC）表型调控的有利分子事件。本研究以钙调素异构体相互转化为分子标记，探讨了平滑肌细胞分化的分子机制。CaD的基因组分析表明，在两个水平，转录和独特的剪接的表达变化的亚型调节，h-和L-CaD的表达调节的两个不同的5 '-剪接位点的选择性选择withn外显子3。我们还发现，信号转导介导的α 1 β 1整合素延缓了培养的SMC的一次去分化，并且α 1 β 1整合素定位于分化的SMC的细胞粘附层。在此培养体系下，h-CaD和其他分子标记如高Mr原核细胞， ...更多信息 肌球蛋白和后黏着斑蛋白在SMC中维持数天。因此，我们利用该培养体系对平滑肌细胞中CaD基因进行了转录因子分析。在原代培养的SMC、小鼠骨骼肌细胞系（C2 C12细胞）和HeLa细胞中的瞬时转染实验显示，CaD启动子活性在SMC中是高水平的，但在其他细胞中是极低的。此外，CaD未分化SMC的启动子活性和蛋白水平均高于去分化SMC。SMC中高水平的启动子活性依赖于位于转录起始位点上游-309至-300处的独特的CArG盒样基序CCAAAAAAGG，该基序与其5 ′-snd 3 ′-侧翼的6个核苷酸序列（CArG 1）一起对于增强启动子活性是必需的。这些结果表明CArG 1是CaD基因高表达的细胞类型特异性的一个必要的顺式元件，CArG 1的功能可能受SMC的表型调控。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