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Transcriptional Regulation of the cardiovaocular genes in respone to stress

应激反应中心血管基因的转录调控

基本信息

批准号：
11470156
负责人：
KURABAYASHI Masahiko
金额：
$ 6.59万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

Despite the pleiotropic effects of TGF-β on vascular smooth muscle cells (VSMCs), only few genes have been characterized as direct targets of TGF-β in VSMCs. CARP, cardiac ankyrin repeat protein, has been thought to be expressed exclusively in the heart. In this study we showed that CARP is expressed in the vasculature after balloon injury and in cultured VSMCs in response to TGF-β. Analysis of a half-life of the cytoplasmic CARP mRNA levels and the transient transfection of the CARP promoter/luciferasere gene indicate that the regulation of CARP expression is increased by TGF-β at the transcriptional level. Transfection of expression vectors encoding Smads significantly activated the CARP promoter/luciferase activity. Deletion analysis and site-specific mutagenesis of the CARP promoter indicate that TGF-β response element is localized to CAGA motif at -108 bp relative to the transcription start site. Electrophoretic mobility shift assays showed that the binding activity to the CAGA motif was increased in nuclear extracts of cultured VSMCs by TGF-β. Cells transfected with adenovirus vector expressing CARP showed a significant decrease in DNA synthesis. Overexpression of CARP enhanced the TGF-β-mediated inhibition of the DNA synthesis. These data indicate that CARP is a downstream target of TGF-β/Smad-signaling in VSMCs, and suggest a role of CARP in mediating the inhibitory effects of TGF-β on the proliferation of VSMCs.

尽管TGF-β对血管平滑肌细胞（VSMC）具有多效性作用，但只有少数基因被表征为VSMC中TGF-β的直接靶点。CARP，心脏锚蛋白重复序列蛋白，被认为只在心脏中表达。在这项研究中，我们发现CARP在球囊损伤后的血管系统中表达，并在培养的VSMC中响应TGF-β。细胞质CARP mRNA水平的半衰期分析和CARP启动子/转录酶基因的瞬时转染表明，CARP表达的调节在转录水平上由TGF-β增加。转染编码Smads的表达载体显著激活CARP启动子/荧光素酶活性。CARP启动子的缺失分析和定点突变表明，TGF-β反应元件定位于相对于转录起始位点的-108 bp处的CAGA基序。电泳迁移率变动分析显示TGF-β可增强培养的VSMCs核提取物与CAGA基序的结合活性。用表达CARP的腺病毒载体转染的细胞显示DNA合成显著减少。过表达CARP可增强TGF-β对DNA合成的抑制作用。这些数据表明，CARP是VSMCs中TGF-β/Smad信号传导的下游靶点，并且表明CARP在介导TGF-β对VSMCs增殖的抑制作用中起作用。