Functional Analysis of p53-Regulatory Protein CARP in Atherosclerosis

转化生长因子-β在血管疾病的发生发展中起重要作用。尽管转化生长因子-β对血管平滑肌细胞(VSMCs)具有多效性，但很少有基因被鉴定为转化生长因子-β的直接靶点。在本研究中，我们发现CARP在球囊损伤后的新生内膜SMC和培养的VSMC对转化生长因子-β的反应中均有表达。转化生长因子-β在转录水平上调了鲤鱼基因的表达。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。转染腺病毒载体的细胞DNA合成明显减少。鲤鱼的过表达增强了转化生长因子-β介导的DNA合成的抑制。这些数据表明，CARP是转化生长因子-β/Smad信号通路的下游靶点，并可能参与介导转化生长因子-β对血管平滑肌细胞增殖的抑制作用。我们还研究了鲤鱼对多能10T1/2细胞和血管SMC中SMC基因表达的影响。结果表明，TGFb1通过增加sRf的表达来诱导依赖Carg的Sm22α基因的表达，而酪氨酸激酶src家族在这一作用中起重要作用。此外，鲤鱼过表达可增强转化生长因子β1诱导的Sm22α基因的表达，而不影响SRF的表达。这些发现揭示了鲤鱼在发育过程中调节SMC基因表达的作用以及在血管疾病中的作用。

TGF-β plays a major role in the development of vascular diseases. Despite the pleiotropic effects of TGF-b on vascular smooth muscle cells (VSMCs), only few genes have been characterized as direct targets of TGF-β in VSMCs. In this study we showed that CARP is expressed in neointimal SMCs after balloon injury and in cultured VSMCs in response to TGF-β. The CARP gene expression is increased by TGF-β at the transcriptional level. Transfection of expression vectors encoding Smads significantly activated the CARP promoter/luciferase activity. Cells transfected with adenovirus vector expressing CARP showed a significant decrease in DNA synthesis. Overexpression of CARP enhanced the TGF-β-mediated the inhibition of the DNA synthesis. These data indicate that CARP is a downstream target of TGF-β/Smad-signalings in VSMCs, and suggest a role for mediating the inhibitory effects of TGF-β on the proliferation of VSMCs. We also investigated the effects of CARP on the SMC gene expression in the pluripotent 10T1/2 cells and in vascular SMC. The results demonstrated that TGFb1 induces CArG-dependent SM22α gene expression via an increase in SRF expression, and Src-family of tyrosine kinase is important for this effect. Furthermore, CARP overexpression enhanced TGF-β1-induced SM22α gene expression without affecting SRF expression. These findings shed light on the role of CARP for modulating SMC gene expression during development and in vascular disease.