Effect Of CaMK?? RNA Interference On Osteoclast Differentiation And Expression Of NFATc1,TRAP And C-Src Gene

Objectives To study the effect of calmodulin-dependent kianse ?(CaMK?)? RNA interference on osteoclastogenesis,bone resorption function and gene expression of downstream molecules including nuclear factor of activated T-cells cytoplasmic 1(NFATc1),cell-sarcoma receptor coactivator(c-Src)and tartrate resistant acid phosphatase(TRAP),and to verify the vital regulating function and mechanism of CaMK?? on osteoclast differentiation.Methods 1 Gene expression profiles of CaMK?? during osteoclast differentiation.Mouse RAW264.7 cells were induced with 50ng/ml receptor activator of nuclear factor-?B ligand(RANKL)for osteoclastogenesis and the cells were harvested 0d,1d,3d and 5d after induction.Real-time PCR,Western blotting and immunofluorescent cytochemistry were used to detect CaMK?? gene expression during osteoclast differentiation.2 The effect of zoledronate on osteoclastogenesis and expression of osteoclast-related genes.Mouse RAW264.7 cells were divided into two groups,group A and B.Both groups were induced with 50ng/ml RANKL for osteoclastogenesis and cells in group B were also treated with 1�10-6 mol/L zoledronate from day 2 for 2 days.At day 5 and 7,cells were harvested and TRAP staining as well as dentin slice resorption lacunae analysis,were performed to evaluate the difference of osteoclastogenesis and their resorption function between the two groups.Real-time PCR,Western-blot and immunofluorescent cytochemistry were also used to examine gene expressions of CaMK??,NFATc1,TRAP and c-Src in the two groups.3 Influence of zoledronarte acid on protein binding of CaMK? and Calmodulin during osteoclast differentiation.Mouse RAW264.7 cells were divided into two groups,group A and B.Both groups were induced with 50ng/ml RANKL for osteoclastogenesis and cells in group B were also treated with 1�10-6 mol/L zoledronate from day 2 for 2 days.At day 5,cells were harvested,Co-IP and reverse Co-IP were used to analyze the protein binding between CaMK ? and Calmodulin.4 The effect of CaMK?? RNA interference on osteoclastogenesis and expression of downstream genes.Three recombinant CaMK?? RNA interference vectors were constructed using lentivirus carrying green fluorescent protein(GFP).RAW264.7 cells were transfected with negative vector to determine the multiplicity of infection(MOI)and transfection efficiency under the optimal transfection titer.Then RAW264.7 cells were transfected with three recombinant interference vectors under the optimal MOI value.Five days later,Real-time PCR and Western-blot were used to detect m RNA and protein level of CaMK? ? in cells transfected with three recombinant vectors during osteoclast differentiation and the recombinant vector withbest interfering efficiency was chosen for following experiments.RAW264.7 cell were divided into 3 groups,group A,B and C.Group A is control group,while group B and C was transfected with negative vector and recombinant interfering vector respectively.Twelve hours after transfection,virus was removed and 50ng/ml RANKL was added to induce cells for osteoclatogenesis.After 5 days induction,cells were harvested,TRAP staining and dentin slice resorption lacunas analysis were performed to determine the differences of osteoclastogenesis and bone resorption function among cells of three groups.Real-time PCR,Western-blot and immunofluorescent cytochemistry were used to examine gene expressions of CaMK?? and its downstream molecules including NFATc1,TRAP and c-Src.Results 1 At day 0,1,3 and 5 during osteoclast differentiation,m RNA level of CaMK?? were 1.067�0.179,1.840�0.070,9.493�0.453 and 30.767�0.573 respectively,and protein level were 494.567�20.121,663.533�38.741,858.600�19.367 and 980.367�23.403 respectively;compared with day 0,m RNA level and protein level at other time points increased significantly in a time-dependent manner(P<0.01),except protein level at day 1,which showed no significant difference(P>0.05).Immunofluorescence assay showed that protein expression of CaMK? ? was weak at day 0 and 1,protein expression increased remarkablely at day 3 and 5 and multi-nucleated osteoclasts were observed.2 Under the treatment of zoledronate,number of multinucleated osteoclasts,number and size of dentin resorption lacunaes in group B were 11.33�1.52,8.66 �2.08 and 5034.35�775.42 ?m2 respectively,which were significantly decreased when compared with those(37.66�5.68,23.00�4.00 and 15042.71�1906.03 ?m2)in group A(P<0.01)and the decrease were 69.91%,62.60% and 66.53% respectively.Zoledronarte acid also down-regulated gene expressions of CaMK?? and downstream molecules including NFATc1,TRAP and c-Src during osteoclast differentiation.When compared with group A,m RNA of CaMK??,NFATc1,TRAP and c-Src in group B were decreased about 44.603%,54.126%,58.942% and 51.546%(P<0.01),and protein level were decreased about 46.127%,36.799%,27.140% and 32.060%(P<0.01),respectively.Similar decreases of protein level in group B were also observed by immunofluorescent cytochemistry.3 Co-IP and reverse Co-IP examination showed that protein binding between CaMK? and Calmodulin in group B decreased about 59.75% and 50.87%(P<0.01)when compared with group A,while total protein of Calmodulin and CaMK? in group B were also significantly decresed and the decrease were about 52.12% and 51.49%(P<0.01)respectively.4 CaMK?? recombinant interfering vectors were successfully constructed in this experiment.MOI value was 30 under optimal viral titer and transfection efficiency was over 80%.Real-time PCR and Western-blot examination demonstrated that best interfering effect was acquired aftertransfection with #3 recombinant vector and the interfering efficiency was upto 78.158% at m RNA level and 62.226% at protein level.Therefore #3 recombinant vector was used for following experiments.After virus transfection,number of multi-nucleated osteoclast,number and size of dentin resorption lacunas in group C(interfering vector)were 10.670�1.52,87.330�1.528 and 4922.000�64.086?m2,respectively,which were significantly lower than those(22.670�1.2528,12.670�2.082 and 0924.330�66.905?m2)in group B(negative vector),and those(26.670�1.528,16.000�1.000 and 11980.000� 70.000?m2)in group A(control group)(P<0.05).There were no significant difference between group A and B(P>0.05).CaMK?? RNA interference downregulated gene expression of CaMK??,NFATc1,TRAP and c-Src.Real-time PCR examination showed that m RNA level of above genes decreased about 79.872%,49.856%,43.649% and 53.567%(P<0.01)in group C when compared with those in group A,while there were no significant differences between group B and group A(P>0.05).Western-blot examination demonstrated that protein expressions of above genes in group C were significantly lower than those in group B and group A(P<0.01),and the decrease of protein level were about 61.70%,54.22%,46.75% and 45.86% respectively,when compared with group B.Immunofluorescent cytochemistry detection also verified the decreasement of protein level of above genes in group C compared with group A and B.Conclusions 1 Gene expression of CaMK?? increased in a time-depended manner during osteoclast differentiation,and to hint the vital regulating function and mechanism of CaMK?? on osteoclast differentiation.2 Zoledronic acid could significantly inhibit osteoclastogenesis and bone resorption,and down-regulate gene expression of CaMK?? and its downstream molecules including NFATc1,TRAP and c-Src,which may mediate zoledronate-induced inhibition of osteoclastogenesis.3 Zoledronic acid could inhibit protein binding between CaMK? and Calmodulin,which may play a vital role for zoledronate-induced inhibition of osteoclastogenesis.4 CaMK?? RNA interference could significantly inhibit ostoclast formation and gene expression of NFATc1,TRAP and cSrc at both m RNA level and protein level,and these genes may play an important role in CaMK?? mediated osteoclast differentiation.