Smad5 Gene In Mouse Embryonic Stem Cell Proliferation, Differentiation And Apoptosis In The Function And Mechanism

Previous studies have shown that Smad5, an important intracellular mediator of transforming growth factor β (TGF-β) family, is required for normal development of the cardiovascular system in vivo. The targeted disruption of SmadS gene resulted in embryonic lethality before gestation day E11.5 due to the multiple embryonic and extraembryonic defects. To further study the function of SmadS gene during the veretebrate development, the Smad5 double knockout embryonic stem (ES) cells differentiation system was employed. We found that the myotubes that differentiated from the homozygous Smad5~ex6/ex6 mutant ES cells underwent collapse and degeneration during the late stages of in vitro differentiation, mimicking the in vivo observation. By electron microscopy, apoptosis and abnormal swollen mitochondria were observed in cardiomyocytes both from Smad5-deficient embryos and from ES-differentiated cells. There was also a significant reduction in mitochondrial membrane potential (△Ψm) and a leakage of cytochrome c from mitochondria into the cytosol of myocytes differentiated from Smad5 mutant ES cells. The expression of p53 was found to be elevated in the differentiated Smad5 mutant myocytes.These results suggest that the Smad5-mediated TGF-P signals may protect cardiomyocytes from apoptosis by maintaining the integrity of the mitochondria, probably through suppression of p53 mediated pathways.We also found that undifferentiated Smad5-deficient ES cells has a higher proliferative potential than wild type cells. So we investigated the effect of apoptosis stimuli to ES cells. We exposure ES cells to DNA-damaging agents, including UV irradiation (p53-dependent) and adriamycin treatment (p53-independent), induce ES cells apoptosis. We found that ES cells lacking Smad5 gene are higher susceptible to apoptosis stimuli and p53, Bcl-2 involvement in the apoptotic response of ES cells.Although we found evelated apoptosis in undifferentiated ES cells and differentiated cardiomyocytes, we want to know that apoptosis take place disorder is the result of changed development of early embryo or the direct effect of lacking SmadS gene.