Calmodulin Kinase Ⅱ Activation Of Mitogen-activated Protein Kinase In PC12 Cell Following All-trans Retinoic Acid Treatment

ObiectiveTo construct RNAi expression vector and investigate its inhibitory effects on CaMKIIβexpression of PC12 cell line.MethodSense and antisense oligonucleotides designed and synthesized were annealed and ligated into siRNA expression vectors. After recombinants transfected into PC12 cell line with liposome, RNA and prtein were extracted in different time. The expression of CaMKIIβwas detected by RT-PCR and Western blot.ResultExpression vectors could reduce the expressions of CaMKIIβmRNA and protein in PC12 cell line, compared with blank vector group, the former, the ratio of inhibition of the expression of CaMKIIβmRNA was17.95%,46.40%,64.69%和49.37%, in 24th, 48th,72th and 96th hour, the ratio of inhibitory of the expression of CaMKIIβprotein was 74.77% in 72th hour. RNAi expression vectors can effectively inhibit the expression of CaMKIIβin PC12 cell line. Objective:To study the role of CaMKII signaling in RA activated ERK and p38, in order to explore the mechanism of RA induced apoptosis, trying to reveal the possible molecular mechanism of RA induced apoptosis of neuronal cells and relation between apoptosis and NTDs.MethodDifferent time points post RA exposure were determined by Western blot analysis. Apoptosis of PC12 were detected by microscopic observation, condensation of nuclear chromatin and DNA Ladder. Furthermore, transient transfection of PC12 cells with siRNA against CaMKII markedly reduced RA phosphorylation of ERK and p38.Result1. RA activates CaMKII, ERK1/2 and p38Time course experiments at 10μM RA stimulated PC12 showed increase in phosphorylation of Thr286 of CaMKII (phospho-CaMKII) after 10 min. with maxima at 20 min. Different time points post RA exposure (0, 30min, 1h and 2h) were determined by Western blot analysis.10μM RA also caused increased phosphorylation of ERK and p38. Time course of phosphorylation of ERK and p38 were described, with maxima (1h, 30min) lagging behind that of phospho-CaMK-II.2. CaMKII is upstream of RA activation of ERK and p38The time course of phosphorylation of ERK and p38 were described, with maxima lagging behind that of phospho-CaMKII. Moreover, PC12 cells were pretreated with siRNA for 72h prior to RA stimulation and subsequent analysis of ERK and p38 phosphorylation. The phosphorylation of ERK and p38 analyzed by western blot.in responses to RA. Furthermore, transient transfection of PC12 cells with siRNA against CaMKII markedly reduced RA phosphorylation of p38 after normalization for transfection. Taken together, these data suggest that CaMKII plays role in moleculating activation of ERK and p38 in PC12 cells3. RA induced apoptosis of PC12The microscopic observation of RA-treated cells indicated that RA treatmentg resulted in the substantial death of PC12 cells in preasence of NGF. Cells become small, wizened, expanding foam. The condensation of nuclear chromatin was demonstrated with Hoechst 33258 staining. Compared with the fluorescence from untreated control cells, the fluorescence in RA treated cells was more intense and punctuate, indicating RA induced condensation of the chromatin material.Apoptosis cell death is often associated with internucleosomal DNA fragmentation resulting in nucleosome-size DNA and multiples therof. DNA fragmentation was assessed agarose gel-electrophoresed soluble DNA extracted from PC12 cells after treatment with RA...