E1A激活基因阻遏子过表达诱导体外培养大鼠平滑肌细胞分化

Overexpression of The Cellular Repressor of E1A-Stimulated-genes Regulate The Rat Primary VSMCs Differentiation In vitro

为探讨E1A激活基因阻遏子蛋白 (CREG)对血管平滑肌细胞表型转换的调控机制 ,应用pRC/CMV hCREG真核表达载体转染体外培养的大鼠血管平滑肌细胞 (VSMCs) ,观察了转染前后细胞的表型改变 .结果发现 ,pRC/CMV hCREG质粒转染后 ,大鼠平滑肌细胞增殖受抑 ,细胞分化标志蛋白SMα actin表达显著增加 .免疫共沉淀分析发现 ,CREG与血清反应因子 (SRF)发生相互作用形成复合体 ,在CREG基因过表达时 ,与CREG结合的SRF蛋白增加 .凝胶迁移阻滞分析 (GSMA)和抗体凝胶迁移阻滞分析 (supershift)显示 ,过表达的CREG蛋白与SRF蛋白共同结合到SMα actin基因启动子区CArG位点上 ,可能参与SMα actin表达的调控 .构建SMα actinpromoter pCAT 3报告基因载体并与pRC/CMV hCREG质粒共转染VSMCs也证实 ,CREG蛋白过表达可增强SMα actin基因表达 .上述研究提示 。

To elucidate the role of cellular repressor of El A-stimulated genes ( CREG) in the phenotype modulation of vascular smooth muscle cells (VSMCs), human CREG (hCREG) was overexpressed in primary rat VSMCs was using a mammalian expression vector pRC/CMV-hCREG. Transient transfection of VSMCs with hCREG inhibited cell proliferation and DNA synthesis as evidenced by decrease in BrdU incorporation. Moreover, CREG overexpression increased SM alpha-actin protein level in VSMCs. Co-transfection of VSMCs with SM alpha-actin promoter reporter gene with hCREG enhanced SM alpha-actin promoter activity, suggesting that elevated SM alpha-actin protein production resulted from increased gene transcription. The mechanism by which CREG regulates VSMC phenotype conversion was further investigated by analysis of interaction of CREG with serum response factor (SRF), an important transcriptional factor involved in VSMC differentiation. CREG co-immunoprecipitated with SRF and CREG overexpression increased the SRF bound to CREG. In addition, gel mobility shift assay and supershift assay showed that CREG bound together with SRF to the CArG site of SM alpha-actin promoter. These results suggest that CREG acts as a transcriptional factor and interacts with SRF to promote VSMC differentiation.