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

OBJECTIVE: To investigate the roles of extracellular signal-regulated kinase 5 (ERK5) and c-Jun N-terminal kinase (JNK) in the differentiation of C3H10T1/2 cells into cardiomyocyte-like cells induced by bone morphogenetic protein 9 (BMP9).

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 BMP9, 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 ERK5, 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.