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.

Caldesmon（CAD）在肌动球蛋白系统中起着至关重要的作用，分布在平滑肌和非肌肉细胞中，并且其高MR形式（H-CAD）和低MR形式（1-CAD）之间的同等相互转换是对平滑肌（SMCS）表型调节的有利分子标记。 CAD基因的表达水平在开发SMC中被上调。我们发现，层粘连蛋白上的原代培养的Gizzard SMC保持分化的表型。这项研究的重点是使用当前培养系统的SMC中的转录机制。在这里，我们表征了CAD基因中SMC型启动子的转录特性，该基因显示了SMC中的高活性。分化和去分化细胞的两种表型中CAT构建体的瞬时转染表明，高水平的CAD启动子活性取决于一个独特的类似CARG盒子的基序CCAAAAAAGG，位于转录起点的AT-309 TO-300。这种类似carg盒的基序类似于血清反应元素（SRE），而CAD基因中的该基序无法在SMC中表现出血清责任。我们还通过凝胶转移测定法确定了一种特定的核蛋白因子与该基序相互作用，并发现该蛋白质因子的量比从分化的SMC中的核提取物中的核提取物中大得多。使用抗神经响应因子（SRF）IgG的凝胶移位测定导致了超弹性复合物，这表明与该carg盒样基序相互作用的蛋白质因子可能是SRF或与SRF相关的核蛋白。 carg盒状基序也位于其他SMC特异性细胞骨架蛋白的启动子区域，例如α-SM肌动蛋白，SM22和alpha1整合蛋白。从这些结果中，SMC特异性的转录调控似乎是通过一种共同的机制来控制的，其中涉及类似CARG盒的基序和SRF或与SRF相关的蛋白质因子。