过刊浏览

AMPK激活通过负性调控C/EBPβ抑制小鼠心脏成纤维细胞TGFβ1表达

肖晗, 朴成实, 陈瑞飞, 张幼怡^*

北京大学第三医院血管医学研究所，卫生部心血管分子生物学与调节肽重点实验室，分子心血管学教育部重点实验室，心血管受体研究北京市重点实验室，北京100191

摘要

腺苷酸活化蛋白激酶(AMP-activated protein kinase, AMPK)激活具有抗纤维化的作用，但其具体机制仍不十分清楚。本研究拟在心脏成纤维细胞中，明确AMPK激活对血管紧张素II (angiotensin II, AngII)引起的转化生长因子β1 (transforming growth factor-β1, TGFβ1)表达的作用及其具体机制。分离培养成年小鼠心脏成纤维细胞，酶联免疫吸附实验检测TGFβ1蛋白表达，免疫印迹实验检测AMPK活性和CCAAT/增强子结合蛋白β (CCAAT/enhancer-binding protein β, C/EBPβ)表达，双荧光素酶报告基因实验检测TGFβ1转录活性。结果显示，给予AMPK激活剂AICAR预处理可以抑制AngII引起的TGFβ1产生增加，这一抑制作用可被AMPK抑制剂Compound C逆转。生物信息学分析显示在小鼠Tgfb1启动子区存在C/EBPβ转录因子可能的结合位点。双荧光素酶报告基因实验表明，对于转染Tgfb1启动子区序列质粒的小鼠胚胎成纤维细胞，AngII可以增加TGFβ1转录活性；但是对于转染缺失C/EBPβ结合位点的Tgfb1启动子区序列质粒的细胞，AngII则不能增加其转录活性，提示 C/EBPβ介导了AngII引起的TGFβ1转录表达。进一步免疫印迹实验显示，AICAR可以抑制AngII引起的C/EBPβ增加。上述结果表明，AMPK激活可以抑制AngII引起的TGFβ1表达增加，其作用机制是通过抑制转录因子C/EBPβ表达，进而抑制TGFβ1表达。该研究结果提示了AMPK抗纤维化作用的一个新机制。

关键词： 腺苷酸活化蛋白激酶; C/EBPβ ; 心脏成纤维细胞 ; 转化生长因子β1

分类号：R363.2

AMP-activated kinase activation inhibits transforming growth factor-β1 production in cardiac fibroblasts via targeting C/EBPβ

XIAO Han, PIAO Cheng-Shi, CHEN Rui-Fei, ZHANG You-Yi^*

Institute of Vascular Medicine, Peking University Third Hospital, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides, Ministry of Health, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education and Beijing Key Laboratory of Cardiovascular Receptors Research, Beijing 100191, China

Abstract

AMP-activated protein kinase (AMPK) activation has been shown to protect against fibrosis. However, the underlying mechanism remains unclear. Here we explored the effect of AMPK activation on transforming growth factor-β1 (TGFβ1) production induced by angiotensin II (AngII) in cardiac fibroblasts and the underlying mechanisms. Adult mouse cardiac fibroblasts were isolated. TGFβ1 and AMPK activity were determined by ELISA and Western blots, respectively. Pretreatment of AMPK activator AICAR inhibited TGFβ1 production induced by AngII in cardiac fibroblasts, which was reversed by AMPK inhibitor compound C. Furthermore, bioinformatics predicted a potential CCAAT/enhancer-binding protein β (C/EBPβ) binding site in the promoter region of the mouse Tgfb1 gene. Luciferase reporter with wild type, but not deleted, C/EBPβ binding sites transfection in mouse embryonic fibroblasts showed increased TGFβ1 transcriptional activity induced by AngII, indicating that C/EBPβ mediates AngII-induced TGFβ1 transcript expression. Pretreatment of AICAR inhibited C/EBPβ expression induced by AngII. In conclusion, AMPK activation inhibited TGFβ1 production induced by AngII in cardiac fibroblasts through targeting C/EBPβ. This finding provides a new mechanism underlying the anti-fibrogenic effects of AMPK activation.

Key words: AMPK; C/EBPβ ; cardiac fibroblast ; TGFβ1

收稿日期：2016-10-12　　录用日期：2016-12-15

通讯作者：张幼怡　　E-mail: zhangyy@bjmu.edu.cn

引用本文：

肖晗, 朴成实, 陈瑞飞, 张幼怡. AMPK激活通过负性调控C/EBPβ抑制小鼠心脏成纤维细胞TGFβ1表达[J]. 生理学报 2017; 69 (2): 123-128.

XIAO Han, PIAO Cheng-Shi, CHEN Rui-Fei, ZHANG You-Yi. AMP-activated kinase activation inhibits transforming growth factor-β1 production in cardiac fibroblasts via targeting C/EBPβ. Acta Physiol Sin 2017; 69 (2): 123-128 (in Chinese with English abstract).