The Role Of CaMKII-TAK1-p38 Signaling Pathway In Apoptosis Of Neural Stem Cells

ObjectiveTo study the role of CaMKII ,TAK1, cytochrome c in RA induced apoptosis of neural stem cells, in order to explore the mechanism of RA induced apoptosis, trying to reveal the possible molecular mechanism of RA induced apoptosis of neural stem cells and relation between apoptosis and NTDs.MethodDifferent time points post RA exposure were determined by Western blot analysis. Furthermore, neural stem cells pretreated with KN-93 for 15 min were treated with RA, apoptosis of neural stem cells were detected by flow cytometry and DNA Ladder,and the phosphorylation of CaMKII,TAK1, p38 were detected by Western blot analysis.Result1. RA activates CaMKII, TAK1 and p38Time course experiments at 16μM RA stimulated neural stem cells showed increase in phosphorylation of Thr286 of CaMKII (phospho-CaMKII) after 5min, with maxima at 10 min. Different time points post RA exposure (0, 5min, 10min,20min,40min and 80min) were determined by Western blot analysis.16μM RA also caused increased phosphorylation of TAK1 and p38. Time course of phosphorylation of TAK1 and p38 were described, with maxima (20min, 40min) lagging behind that of phospho-CaMKII.2. RA activates CaMKII-TAK1-p38 Signaling PathwayThe time course of phosphorylation of TAK1 and p38 were described, with maxima lagging behind that of phospho-CaMKII. Moreover,neural stem cells were pretreated with KN-93 for 15 min prior to RA stimulation and subsequent analysis of TAK1 and p38 phosphorylation. Furthermore, pretreated with KN-93 for neural stem cells against CaMKII markedly reduced RA phosphorylation of TAK1 and p38 . Taken together, these data suggest that RA activates CaMKII-TAK1-p38 Signaling Pathway.3. CaMKII activated by RA induced apoptosis of neural stem cells initiates mitochondria-mediated pathway of apoptosis.neural stem cells pretreated with KN-93 for 15 min were treated with RA, apoptosis of neural stem cells were detected by flow cytometry and DNA Ladder.The data showed that pretreated with KN-93 for neural stem cells against CaMKII markedly reduced RA induced apoptosis.we identified the release of apoptosis cells mitochondria cytochrome c by Western-blotting. The results showed that after treatment of neural stem cells by RA, apoptosis factor cytochrome c was released. In conclusion ,these data suggest that CaMKII activated by RA induced apoptosis of neural stem cells initiates mitochondria-mediated pathway of apoptosis.