ERK5信号通路参与BMP9诱导C3H10T1/2细胞向心肌样细胞的分化

Involvement of ERK5 signaling pathway in the cardiomyocyte-like differentiation of C3H10T1/2 cells induced by bone morphogenetic protein 9

摘要:

目的：研究细胞外信号调节激酶5（extracellular signal-regulated kinase 5，ERK5）信号通路在骨形态发生蛋白9（bone morphogenetic protein 9，BMP9）诱导C3H10T1/2细胞向心肌样细胞分化中的作用。方法：用AdBMP9和AdGFP转染C3H10T1/2细胞，流式细胞术检测转染效率，Western blot检测磷酸化ERK5（phosphorylation ERK5，p-ERK5）和ERK5的表达水平，免疫荧光检测p-ERK5在细胞内的表达位置；ERK5特异性抑制剂BIX02189预处理细胞再经AdBMP9诱导，Western blot检测p-ERK5和ERK5表达水平，RT-qPCR检测心肌特异性基因肌细胞增强因子（myocyte enhancer factor，MEF）2C、GATA结合蛋白4（GATA binding protein 4，GATA4）表达，Western blot检测心肌特异性蛋白缝隙连接蛋白４３（connexin 43，CX43）、心肌肌钙蛋白T（cardiac troponin T，cTnT）表达。结果：AdGFP与AdBMP9的转染效率达50%左右，经AdBMP9诱导后的细胞p-ERK5表达明显增高（P＜0.01），免疫荧光结果显示BMP9组p-ERK5表达明显增强且主要集中于胞核；15 ?滋mol/L BIX02189可完全抑制p-ERK5的表达（P＜0.01），且明显降低了由AdBMP9诱导的C3H10T1/2细胞GATA4、MEF2C基因与CX43、cTnT蛋白表达（P=0.000）。结论：BMP9可以通过激活ERK5信号通路调控C3H10T1/2细胞向心肌样细胞分化。

Abstract:

Objective：To investigate the role of ERK5 signal pathway in the differentiation of C3H10T1/2 cells into cardiomyocyte-like cells induced by bone morphogenetic protein 9（BMP9）. Methods：C3H10T1/2 cells were infected with recombinant adenovirus AdBMP9 and AdGFP，and the transfection efficiency was detected by flow cytometry. Expression levels of phosphorylated ERK5（p-ERK5） and ERK5 were detected by Western blot and the expression sites of p-ERK5 in the cell were detected by immunofluores－cence. C3H10T1/2 cells were preprocessed with ERK5 special inhibitor BIX02189 and induced by AdBMP9. Expression levels of p-ERK5 and ERK5 were detected by Western blot；expression levels of myocardial specificity gene myocyte enhancer factor 2C（MEF2C） and GATA4 were measured by real-time fluorescence quantitative PCR and expression levels of myocardial specificity protein connexin 43（CX43） and cardiac troponin T（cTnT） were detected by Western blot. Results：The transfection efficiencies of AdGFP and AdBMP9 were about 50% and the expression level of p-ERK5 after being induced by AdBMP9 was significantly higher（P<0.01）. Immunofluorescence showed that the expression of p-ERK5 of BMP9 group was obviously increased and mainly concen－trated in the nucleus. 15 ?滋mol/L BIX02189 completely inhibited the expression of p-ERK5（P<0.01） and significantly inhibited the AdBMP9 induced expression of MEF2C，GATA4 genes and CX43，cTnT proteins in C3H10T1/2 cells（P=0.000）. Conclusion：The ERK5 signal pathway can be activated by BMP9 to promote cardiomyocyte-like differentiation from C3H10T1/2 cells.