Transcriptional Regulation of the cardiovaocular genes in respone to stress

尽管转化生长因子-β对血管平滑肌细胞(VSMCs)具有多效性，但在VSMC中，很少有基因被确定为转化生长因子-β的直接靶点。CARP，心脏锚蛋白重复蛋白，被认为只在心脏中表达。在这项研究中，我们发现，在球囊损伤后的血管系统和培养的血管内皮细胞对转化生长因子-β的反应中，CARP有表达。对鲤鱼胞质基因半衰期和鲤鱼启动子/荧光素酶基因瞬时转染的分析表明，转化生长因子-β在转录水平上促进了鲤鱼基因的表达调控。转染编码Smads的表达载体显著激活了鲤鱼启动子/荧光素酶的活性。对鲤鱼启动子的缺失分析和定点突变表明，转化生长因子-β反应元件定位于cagA基序的转录起始点的-108bp处。凝胶迁移率改变分析表明，转化生长因子-β使培养的VSMC核提取液与cagA基序的结合活性增强。转染腺病毒载体的细胞DNA合成明显减少。CARP的过表达增强了转化生长因子-β对DNA合成的抑制作用。这些结果表明，CARP是转化生长因子-β/Smad信号转导通路的下游靶点，在介导转化生长因子-β抑制血管平滑肌细胞增殖中起着重要作用。

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.