国家自然科学基金资助项目 (30570661), 国家科技部基础研究重大项目前期研究专项资助项目(2005CCA03100).
This work was supported by grants from The National Natural Science Foundation of China (30570661), Special Found for Preliminary Research of Key Basic Research Project of Ministry of Science and Technology of China (2005CCA03100).
血管紧张素Ⅱ (AngⅡ) 可诱导其前体基因在血管平滑肌细胞 (vascular smooth muscle cells,VSMC) 中进行表达,其作用机制与促进转录激活蛋白-1 (activating protein-1,AP-1) 的基因调控区中存在的AP-1位点结合有关. 为进一步明确AngⅡ调节AP-1结合活性的分子机制,用放线菌酮 (cycloheximide,CHX) 作为c-Jun磷酸化抑制剂,经DNA-蛋白质相互作用和蛋白质印迹实验,探讨AngⅡ对AP-1结合活性的影响并探讨其分子机制. 结果表明,受AngⅡ刺激的VSMC,其核蛋白中AP-1的组成亚基之一c-Jun水平明显升高. 免疫细胞化学染色显示,在被AngⅡ处理的细胞中,c-Jun主要定位于细胞核,胞浆中几乎检测不出该转录激活蛋白的存在. 用丝氨酸磷酸化抗体检测证实,AngⅡ可诱导c-Jun磷酸化. 电泳迁移率改变分析(electrophoretic mobility shift assay,EMSA)显示,c-Jun的磷酸化水平与AP-1结合血管紧张素原基因顺式元件的活性,和对该基因的转录激活作用呈正相关关系,CHX通过阻断c-Jun磷酸化抑制AngⅡ 诱导的AP-1结合活性,但是不影响c-Jun的表达水平. 上述结果提示,AP-1的磷酸化活化是AngⅡ正反馈调节其前体基因表达的重要机制之一,首次发现CHX是c-Jun磷酸化的抑制剂.
Angiotensin Ⅱ (AngⅡ) can induce the expression of its precursor, angiotensinogen, in vascular smooth muscle cells (VSMC), which is related with increased activating protein-1 (AP-1) binding to its cis-element located in the angiotensinogen gene promoter. In the present study, cycloheximide (CHX) was used as an inhibitor to interrupt c-Jun, the role of AP-1 in AngⅡ-induced its precursor gene activation was investigated by DNA-protein interaction and immunoblotting. The results showed that the level of c-Jun, the component of transcription factor AP-1, was significantly increased in the nucleus of VSMC after AngⅡ treatment. The majority of c-Jun was found in the nucleus but hardly detected in the cytoplasm by immunocytochemistry staining. Immunoprecipitation assays confirmed that AngⅡ could induce serine phosphorylation of c-Jun. EMSA results indicated that the level of phosphorylated of c-Jun had a positive correlation with AP-1 binding activity to cis-acting element of angiotensinogen gene and transcription activation of angiotensinogen. CHX inhibited AngⅡ- induced binding activity of AP-1 by reducing the phosphorylation of c-Jun, though it did not affect the expression of c-Jun. These findings suggest that the AP-1 phosphorylation induced by AngⅡ is one of the important mechanisms whereby AngⅡ regulates its precursor gene expression in feedback manner. It is found that CHX is an inhibitor to phosphorylation of c-Jun.
李爱英,温进坤,韩梅. AP-1在AngⅡ正反馈调节其前体基因表达中的作用[J].生物化学与生物物理进展,2006,33(8):775-780
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