Transcriptional Regulation of the cardiovaocular genes in respone to stress

尽管TGF-β对血管平滑肌细胞（VSMCs）有多种作用，但只有少数基因被认为是TGF-β在VSMCs中的直接靶点。CARP，心脏锚蛋白重复序列蛋白，一直被认为只在心脏中表达。在本研究中，我们发现在球囊损伤后的血管和培养的VSMCs中，鲤鱼在TGF-β的作用下表达。细胞质CARP mRNA半衰期水平分析和瞬时转染CARP启动子/荧光素酶基因表明，TGF-β在转录水平上增加了对CARP表达的调控。转染编码Smads的表达载体可显著激活CARP启动子/荧光素酶活性。CARP启动子的缺失分析和位点特异性突变表明，TGF-β应答元件定位于相对于转录起始位点-108 bp的CAGA基序上。电泳迁移率转移实验表明，TGF-β可提高培养的VSMCs核提取物与CAGA基序的结合活性。转染表达CARP的腺病毒载体后，细胞的DNA合成明显减少。过表达CARP可增强TGF-β介导的DNA合成抑制。这些数据表明，CARP是TGF-β/ smad信号在VSMCs中的下游靶点，并提示CARP在介导TGF-β对VSMCs增殖的抑制作用中起作用。

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.