Transcriptional regulation of caldesmon gene in smooth muscle cells

Caldesmon （CaD）在肌动球蛋白系统中起着至关重要的作用，分布于平滑肌细胞和非肌肉细胞中，其在高Mr形式（h-CaD）和低Mr形式（1-CaD）之间的异构转换是平滑肌细胞（SMCs）表型调节的有利分子标记。CaD基因的表达水平在发育中的SMCs中上调。我们发现原代培养的砂囊SMCs在层粘连蛋白作用下保持分化表型。本研究的重点是利用现有的培养系统研究SMCs的转录机制。在这里，我们描述了CaD基因中smc型启动子的转录调控，该基因在SMCs中显示出高活性。在两种表型的SMCs、分化细胞和去分化细胞中瞬时转染CAT构建物，发现高水平的CaD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 -300处。这个CArG盒状基序与血清反应元件（SRE）相似，而CaD基因中的这个基序在SMCs中不能表现出血清责任。我们还通过凝胶移位实验确定了与该基序相互作用的特定核蛋白因子，并发现分化的SMCs的核提取物中该蛋白因子的含量远高于未分化的SMCs。使用抗血清反应因子（SRF） IgG进行凝胶移位实验，发现一个超移位复合物，表明与这个CArG盒状基序相互作用的蛋白因子可能是SRF或SRF相关的核蛋白。CArG盒状基序也定位于其他smc特异性细胞骨架蛋白的启动子区域，如α - sm肌动蛋白、SM22和α 1整合素。从这些结果来看，smc特异性转录调控似乎是由一种共同的机制控制的，其中包括CArG盒状基序和SRF或SRF相关蛋白因子。

Caldesmon (CaD) , which plays a vital role in the actomyosin system, is distributed in smooth muscle and nonmuscle cells, and its isoformal interconversion between high Mr form (h-CaD) and low Mr form (1-CaD) is a favorable molecular marker for phenotypic modulation of smooth muscle cells (SMCs). The expressional level of CaD gene is up-regulated in developing SMCs. We found that primary cultured gizzard SMCs on laminin maintain differentiated phenotype. This study has focused on the transcriptional mechanism in SMCs using the present culture syetem. Here, we have characterized the transcriptional tegulation of the SMC-type promoter in CaD gene which shows the high activity in SMCs. Transient transfection of CAT constructs in both phenotypes of SMCs, differentiated and dedifferentiated cells, revealed that the high level of CaD promoter activity depends on a unique CArG box-like motif, CCAAAAAAGG,located at-309 to-300 upstream from the transcriptional starting site. This CArG box-like motif is similar to serum response element (SRE) , while this motif in the CaD gene is not able to exhibit serum responsibility in SMCs. We also identified a specific nuclear protein factor interacting with this motif by gel shift assay, and found that the amount of this protein factor is much higer in the nuclear extracts from differentiated SMCs than those from dedifferentiated SMCs. Gel shift assay using anti-serum response factor (SRF) IgG resulted in a supershifted complex, suggesting that the protein factor interacting with this CArG box-like motif might be SRF or SRF-related nuclear protein. CArG box-like motif are also localized in the promoter regions of other SMC-specific cytoskeletal proteins, such as alpha-SM actin, SM22, and alpha1 integrin. From these results, SMC-specific transcriptional regulation would seem to be controled by a common mechanism, in which CArG box-like motif and SRF or SRF-related protein factor are involved.