Effect Of CaMKⅡδ Gene Silencing On C-fos,c-jun,CREB Expression And Osteoclast Differentiation

Objectives To study the effect of calmodulin-dependent kinase Ⅱ(CaMKⅡδ)gene silencing on osteoclasts differentiation,bone resorption function and gene expression of c-fos,c-jun and CREB during osteoclast differentiation,to explore the role of these genes in CaMKⅡδ-regulated osteoclast differentiation and reveal the molecular mechanism of osteoclast differentiation regulation.Methods 1.Determination of efficiency of CaMKⅡδ RNA interference.CaMKⅡδ RNA interference vector was constructed with lentivirus.RAW264.7 cells,a pre-osteoclast cell line,were used in this study and divided into three groups,the blank group,negative vector group and interference vector group.The cells in latter two groups were transfected wtih negative virus vector and recombinant interference vector,respectively.Twelve hours after transfection,the cells were cultured in fresh medium and treated with 50ng/ml nuclear factor-KB receptor activator ligand(RANKL)for osteoclastic induction.Five days later,the cells were harvested and Real-time PCR and Western blotting were used to detect mRNA and protein expression levels of CaMKⅡδ to determine the interference efficiency.2.Effect of CaMKⅡδ gene silencing on osteoclast differentiation and bone resorption function.RAW264.7 cells were transfected with negative and interference virus as described previously,induced with RANKL and harvested 5 days later.The effect of silencing CAMKⅡδ gene on osteoclast differentiation and bone resorption function was detected by TRAP detection and bovine bone resorption lacuna analysis,respectively.3.The effect of CaMKⅡδ gene silencing on gene expression of c-fos,c-jun and CREB.The cells in three groups were treated with RANKL for 2 days and then harvested.The expression of c-fos,c-jun and CREB genes were detected by Real-time PCR and Western-blot.4.The effect of CaMKⅡδ gene silencing on expression of c-fos,c-jun and CREB proteins detected by fluorescent cytochemistry.The cells were treated as previously described and harvested 3 days later,then immunofluorescence cytochemistry was performed to evaluate protein expression of the three genes.Results 1.Real-time PCR examination showed that the relative expression levels of CaMKⅡδ mRNA in the blank group,the negative vector group and the interference vector group were 1.282±0.234,0.950±0.275 and 0.377±0.148,respectively(P<0.05).There was no significant difference for CaMKⅡδ mRNA between the blank group and negative vector group(P>0.05),while CaMKⅡδ mRNA in interference vector group decreased 70.6%when compared with blank group(P<0.01).The result indicate that the interference efficiency at mRNA level was 70.6%.Western-blotting examination showed that the relative expression levels of CaMKⅡδ protein(ratio to β-actin)in the blank group,the negative vector group and the interference vector group were 1.063±0.457,1.006±0.139 and 0.295±0.032,respectively.No significant difference was observed between the first two groups(P>0.05),whereas the CaMKⅡδ protein level in the interference vector group decreased 72.2%when compared with the blank group(P<0.01).Therefore the interference efficiency of CaMKⅡδ was 70.6%at protein level.2.After RANKL induction for five days,the changes of RAW264.7 cells in blank group,negative vector group and interference vector group were observed and TRAP-positive multinucleated osteoclasts were found in all three groups.The number of multinucleated osteoclasts was 12.7±2.49 in blank group,11±1.63 in negative vector group,and 4±1.63 in the interference vector group.Statistical analysis showed that there was no significant difference for osteoclast number between the blank group and the negative vector group(P>0.05),whereas number of osteoclasts in interference vector group decreased significantly when compared with the blank group and the the negative vector group(P<0.05).The size of osteoclasts in the three groups was also evaluated under the microscope and it was found that the size of osteoclasts in the blank group and the negative vector group was large,and the size of multinucleated osteoclasts in the interference vector group was smaller than the other two groups.Formation of bone resorption lacunae on bovine bone slice in three groups was also examined by scanning electron microscopy.The results showed that the number and area of bone absorption lacunae were 10±2.65 and 5694±536.21μm2 respectively in the blank group,12±1.23 and 5247±635.24μm2 respectively in the negative vector group,and 3±2.31 and 2437±143.65μm2 respectively in the interference vector group.Statistical analysis showed that no significant difference in the number and area of bone resorption lacunae was observed between the blank group and the negative vector group(P>0.05),whereas the number and area of bone resorption lacunae in the interference group decreased significantly when compared with the blank group and the interference vector group(P<0.05).Above results indicted that under CaMKⅡδ gene silencing,osteoclasts differentiation and bone resorption function was significantly inhibited.3.CaMKⅡδ gene silencing significantly down-regulated the mRNA levels of c-fos,c-jun and CREB in preosteoclasts and the decrease was 74%,49%and 24%,respectively(P<0.05).CaMKⅡδ gene silencing also significantly reduced the protein levels of c-fos,c-jun and CREB,and the down-regulation were 74.3%,61.3%and 59.2%,respectively.4.Immunofluorescence cytochemistry was performed to evaluate the fluorescent intensity of c-fos,c-jun and CREB proteins in cells of three groups.The results showed that the fluorescent intensity of c-fos,c-jun and CREB proteins was strong both in the blank group and negative vector group,and the proteins were mainly distributed in cytoplasm and partly observed in nuclei of some cells.By contrast,protein fluorescent intensity of the three genes in the inerference group was weak and the proteins were mainly observed in cytoplasm rather than in the nuclei.Above results suggested that CaMKⅡδ gene silencing not only inhibited protein expression of the three genes but also block their function of gene regulation.Conclusions 1.CaMKⅡδ gene silencing could significantly inhibit osteoclast different iation and bone resorption.2.CaMKⅡδ gene silencing significantly inhibited the expression of c-fos,c-jun,CREB mRNA and protein levels and these genes may play a role in the regulation of osteoclast differentiation by CaMKⅡδ.Figure9;Table4;Reference 129...