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Transcriptional regulation of caldesmon gene in smooth muscle cells

平滑肌细胞caldesmon基因的转录调控

基本信息

批准号：
06836011
负责人：
HAYASHI Kenichiro
金额：
$ 1.34万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

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.

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，其高分子量形式（h-CaD）和低分子量形式（1-CaD）之间的异构体转换是平滑肌细胞表型调节的有利分子标记。CaD基因在平滑肌细胞发育过程中表达上调。我们发现原代培养的肌胃平滑肌细胞在层粘连蛋白上保持分化的表型。本研究利用现有的培养系统对SMCs的转录机制进行了研究。在这里，我们已经确定了在SMC中表现出高活性的CaD基因的SMC型启动子的转录tegulation。CAT构建体在两种表型的SMC，分化和去分化细胞的瞬时转染，揭示了高水平的CaD启动子活性依赖于一个独特的CArG盒样基序，CCAAAAAAGG，位于-309至-300上游的转录起始位点。该CArG盒样基序类似于血清反应元件（SRE），而CaD基因中的该基序不能在SMC中表现出血清反应性。我们还通过凝胶迁移实验鉴定了一种与该基序相互作用的特异性核蛋白因子，并发现这种蛋白因子在分化的SMC的核提取物中的含量比去分化的SMC高得多。用抗血清反应因子（SRF）IgG进行凝胶位移实验，结果显示与CArG盒样结构相互作用的蛋白因子可能是SRF或SRF相关的核蛋白。CArG盒样基序也定位于其他SMC特异性细胞骨架蛋白的启动子区，如α-SM肌动蛋白、SM 22和α 1整联蛋白。这些结果表明SMC特异性转录调控可能受一个共同的机制控制，其中涉及CArG盒样基序和SRF或SRF相关蛋白因子。