Involvement Of ERK5and JNK In The Cardiomyocyte-like Differentiation Of C3H10T1/2Cells Induced By BMP9

PART ONE: THE ACTIVATION SITUATION OF JNK ANDERK5OF C3H10T1/2CELLS INFECTED BY BMP9Objective: Detected the activation of JNK and ERK5of C3H10T1/2cells after infected by high-titer adenovirus carried with GFP and BMP9genes.Methods: HEK293cells were cultured and used to repeatedlyamplified and collected high-titer of AdBMP9and AdGFP adenovirus.When the C3H10T1/2cells were affected by high-titer AdBMP9andAdGFP after24hours, the expression of GFP were observed byfluorescence microscope, the transfection efficiency were detected byflowcytometry and the localization of p-JNK and p-ERK5expressed incells were observed by immunofluorescence. The effect of specificinhibitors on cell’s growth was detected by CCK-8, and the activationsituation of JNK and ERK5were analysised by Western blot after cellswere cultured by JNK or ERK5signaling pathway specific inhibitorSP600125or BIX02189. Results:1. C3H10T1/2cells were transfected by the high-titer ofAdBMP9and AdGFP24hours later, the transfection efficiency detectedby flowcytometry reached to about50%.2. The morphology of cells after being transfected by AdBMP9observed by inverted microscope was shifted from polygon to longfusiform with the culture time extended gradually and closer linksbetween cells were formed.3. Immunofluorescence showed that p-JNK and p-ERK5wereexpressed in cells’cytoplasm and nucleus of all groups, but the expressionof p-JNK and p-ERK5was significantly enhanced after induced byAdBMP9, and focused on the nucleus and the most of cytoplasm aroundthe nucleus.4. C3H10T1/2cell’s survival rate were gradually decreased withinhibitor concentration’s increased showed by CCK-8, but concentrationsthat inhibited activation of JNK and ERK5were far below the inhibitor’sIC50, so the inhibition on cell proliferation of inhibitors could negligible.5. The expression of p-ERK5and p-JNK were significantly increaseddetected by Western blot,while the activation rate were decreased with theinhibitor concentration increased.Conclusion: High-titer of adenovirus amplified by HEK293cellsinfected C3H10T1/2cells with higher efficiency, the expression of p-ERK5and p-JNK after C3H10T1/2cells being transfected were significantly increased, and the activation rate decreased with the inhibitor concentrationincreased,15μmol/L BIX02189and20μmol/L SP600125can block theactivation of ERK5and JNK of C3H10T1/2cells induced by AdBMP9. PART TWO: THE DIFFERENTIATED SITUATION OFC3H10T1/2CELLS INTO CARDIOMYOCYTE-LIKECELLS INDUCED BY BMP9AFTER SPECIFICINHIBITION OF JNK AND ERK5Objective:The situations of C3H10T1/2cells differentiationed intocardiomyocyte-like cells induced by AdBMP9after specific inhibited byJNK specific inhibitor SP600125and ERK5specific inhibitor BIX02189.Methods：Use15μmol/L BIX02189,20μmol/L SP600125blockedthe activation of ERK5and JNK, RT-qPCR was performed to analysis theexpression of cardiac-specific genes GATA4, MEF2C of C3H10T1/2cellsafter induced by BMP9one week, Western blot was conducted to measurethe expression of cardiac-specific proteins cTnT and CX43of C3H10T1/2cells after induced by BMP9three weeks, the position of cTnT, CX43incells were observed by immunofluorescence.Results：After BIX02189inhibited the activated of ERK5，theexpression levels of myocardial differentiation markers MEF2C, GATA4,CX43, cTnT of C3H10T1/2cells were significantly suppressed. JNKspecific inhibitor SP600125also inhibited the expression levels of MEF2C,GATA4, CX43, cTnT，but the inhibition of MEF2C and GATA4was not asnotable as that of BIX01289.Conclusion: The excessive activation of ERK5and JNK plays an important role in the differentiation of C3H10T1/2cells intocardiomyocyte-like cells induced by BMP9.