ERK5和JNK参与BMP9诱导的C3H10T1/2细胞向心肌样细胞分化

Involvement of ERK5 and JNK in the BMP9-induced differentiation of C3H10T1/2 cells into cardiomyocyte-like cells

目的研究细胞外信号调节激酶5(ERK5)和c-Jun N端激酶(JNK)在骨形态发生蛋白9(BMP9)诱导的C3H10T1/2细胞向心肌样细胞分化中的作用。方法利用重组腺病毒将BMP9基因导入C3H10T1/2细胞,Western blot法检测加入BMP9及不同浓度ERK5特异性抑制剂BIX02189或JNK特异性抑制剂SP600125后ERK5和JNK的激活情况;实时定量PCR(qRT-PCR)检测BMP9诱导1周后心肌特异性基因肌细胞增强因子2C(MEF2C)、GATA结合蛋白4(GATA4)表达;Western blot法检测经BMP9诱导3周后心肌特异性蛋白缝隙连接蛋白43(CX43)、心肌肌钙蛋白T(cTnT)表达和免疫荧光技术检测CX43、cTnT在细胞内的表达。结果在转染效率达50%左右的情况下,BMP9可过度激活ERK5和JNK,使其磷酸化水平显著增高(P<0.05);BIX02189抑制ERK5激活后,BMP9诱导的C3H10T1/2细胞的心肌分化标志物MEF2C、GATA4、CX43、cTnT均受到显著抑制(P<0.05),JNK特异性抑制剂SP600125也可抑制MEF2C、GATA4、CX43、cTnT表达,但对MEF2C及GATA4的抑制不如BIX02189显著(P<0.05)。结论 ERK5和JNK的过度激活参与了BMP9诱导的C3H10T1/2细胞向心肌样细胞的分化。

Objective To investigate the roles of extracellular signal-regulated kinase 5 （ERKS） and c-Jun N-terminal kinase （JNK） in the differentiation of C3H10T1/2 cells into cardiomyocyte-like cells induced by bone morphogenetic protein 9 （BMPg）. Methods BMP9 gene was imported into C3H10T1/2 cells by recombinant adenovirus. Western blotting was used to detect the actived levels of ERK5 and JNK after cultivated with BMP9 and different concentrations of ERK5 specific inhibitor BIX02189 or JNK specific inhibitor SP600125; real-time quantitative PCR （qRT-PCR） was performed to analyze the expressions of myocardium specific genes GATA binding protein 4 （ GATA4）, myocyte enhancer factor 2C （MEF2C） after one week induced by BMP9; Western blotting was conducted to measure the expressions of myocardium specific proteins connexin 43 （CX43）, cardiac troponin T （cTnT） after three weeks induced by BMPg, and immunofluorescence to observe the positions of CX43 and cTnT in the cells. Results In the case of transfection efficiency up to 50%, BMP9 exceedingly activated ERK5 and JNK, and significantly increased their phosphorylation level （P 〈 0.05 ）. After BIX02189 inhibited the activity of ERKS, the expression levels of myocardial differentiation markers MEF2C, GATA4, CX43, cTnT of C3H10T1/2 cells were significantly suppressed （P 〈 0.05 ）; JNK specific inhibitor SP600125 also inhibited the expression levels of MEF2C, GATA4, CX43, cTnT, but the inhibition on MEF2C and GATA4 was not as notable as that of BIX02189 （P〈0.05）. Conclusion The excessive activation of ERK5 and JNK plays an important role in the differentiation of C3H10T1/2 cells into cardiomyocyte-like cells induced by BMP9.