| Objectives This study is aimed to investigate the influence of Ca2+/Calmodulin dependent kinase II(Ca MKII)delta gene silence by RNA interference on osteoclast differentiation and activity of mitogen-activated protein kinases(MAPKs),c AMP response element binding protein(CREB)and Nuclear factor of activated T-cells cytoplasmic 1(NFATc1),elucidate the mechanism of osteoclast differentiation.Methods 1 The effect and mechanism of CaMKII δ RNA interference on osteoclast differentiation and function.RAW264.7 cells,a mice leukemia macrophage monocyte line,were divided into three groups: control group,negative vector group and interference group.Negative virus vector with green fluorecent protein(GFP)was used to transfect RAW264.7 cells to determine the most suitable MOI and transfection efficiency.Virus transfection lasted for 12 h and then the cells were cultured with new media under the induction of 50ng/ml Ligand of Receptor Activator of Nuclear Factor κB(RANKL)for osteoclast differentiation.Five days later,the cells were harvested for various examinations.the effect of Ca MKII δ RNA interference on osteoclastogenesis and function of bone absorption were examined by tartrate-resistant acid phosphatase(TRAP)staining and bovine bone absorption lacunae analysis.Real-time PCR and Western blot were performed to confirm the effect of Ca MKII δ RNA interference on Ca MKII δ gene expression.Immunofluorescence cytochemistry and Western blot,were also used repectively to explore the expression profile of Nuclear factor of activated Tcells cytoplasmic 1(NFATc1).2 The effect of Ca MKII δ RNA interference on protein activity of CREB.RAW264.7 cells were divided into two groups,negative vector group and interference group.Three days after transfection with virus,cells in both groups were induced with 50ng/ml RANKL for 0h,1h,3h,6h,12 h and 24 h.Western blot was used to identify the level of phosphorylated CREB.3 The effect of Ca MKII δ RNA interference on activity of MAPKs pathway.RAW264.7 cells were divided into two groups,negative vector group and interference group.After virus transfection,the culture media were changed and cells in both groups were induced with 50ng/ml RANKL for 0 min,5 min,10 min,15 min,30 min and 60 min.Western blot was used to determine the level of phosphorylated extracellular signal-regulated kinase 1/2(ERK1/2),c-Jun N-terminal kinase(JNK)and P38 MAPK(P38).4 The effect of MAPKs signal inhibition on NFATc1 expression in osteoclasts.RAW264.7 cells were divided into four groups: control group,ERK1/2 inhibitor(PD98059)group,JNK inhibitor(SP600125)group and P38 inhibitor(SB203580)group.RAW264.7 cells were induced with RANKL for osteoclast differentiation for 3 days and treated with above inhibitors respectively.Expression level of NFATc1 was detected by Western blot and osteoclastogenesis was evaluated by TRAP staining.Results 1 After transfection with negative virus vector,fluorescent miceoscope observation showed that the most suitable MOI value for transfection was 30 and the transfection efficiency was 74.9%.RAW264.7 cells in three groups were successfully differentiated into multinuclear osteoclasts(more than three nucleus)after induction with RANKL.The number of TRAP positive multinuclear osteoclasts,number and size of absorption lacunae on bone slice were 10.8±2.1,8.5±1.1 and 5463±884.5μm2 respectively in CamkⅡ δ RNA interference group,which were significantly lower than those in negative vector group(24.7±2.2,24.8±1.9 and 10491±635.3 μm2 respectively)(p<0.05)and those in control group(27.6±3.9,25.8±2.3 and 11447±710.4 μm2)(p<0.05).Whereas there are no significant difference for above indice between control group and negative vector group(P>0.05).Real-time PCR revealed that CamkⅡ δ m RNA was significantly down-regulated by RNA interference and the interference efficiency was up to 77.2%.Western blot also indicated remarkable decrease of Ca MKII δ protein level in interference group and its protein level was only about 0.33±0.04,which was significantly lower than that(1.11±0.12)in negative vector group(P<0.01).Immunofluorescent examination showed that fluorescent intensity of NFATc1 in CamkⅡ δ RNA interference group was also decreased when compared with those in control group and negative vector group.The decrease of protein level of NFATc1 was also supported by Western-blot and the protein level(1.52±0.04)in CamkⅡ δ RNA interference group was down-regulated about 72.3% when compared with that(2.25±0.12)in negative vector group(P<0.01).2 After virus transfection and RANKL treatment,Western blot indicated that level of phosphorylated CREB was significantly decreased in interference group whem compared with negative vector group and the decrease was about 21%~56%(P<0.05).3 After virus transfection and RANKL treatment,Western blot showed that levels of p-ERK1/2,p-JNK and p-P38 were also significantly decreased when compared with negative vector group and the decreases was about 55%~64% for p-ERK(P<0.05),12%~40% for p-JNK(P<0.01)and 25%~52% for p-P38(P<0.01),respectively.4 In vitro MAPKs signal regulating experiment indicated that three inhibitors,PD98056(ERK1/2 inhibitor),SP600125(JNK inhibitor)and SB203580(P38 inhibitor),all significantly down-regualted protein expression of NFATc1 during osteoclast differentiation and the dereases of NFATc1 protein level in PD98056 group,SP600125 group and SB203580 group were 37.3%(P<0.01),42.1%(P<0.05)and 38.2%(P<0.05)respectively,when compared with control vector group.Among three groups above,number of TRAP positive multinuclear osteoclasts were 9.7±1.5,10.0±2.0 and 8.7±2.1 respectively,which were significantly lower than that(16.0±1.0)in control group,and the decreases was about 39.4% for ERK1/2 inhibitor,37.5% for JNK inhibitor and 45.6% for P38 inhibitor(P<0.01)respectively.Conclusions CamkⅡ δ RNA interference could significantly decrease osteoclast differentiation and CREB,MAPKs(including ERK1/2,JNK,P38)signals may participate in Ca MKII δ mediated osteoclast differentiation. |
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