Effects Of CaMKⅡδ Overexpression On Osteoclast Differentiation And Activity Of Downstream Signaling Molecules Including CREB,ERK,JNK And P38

Objectives This study is aimed to investigate effects of Ca2+/Calmodulin dependent kinase IIδ(CaMKⅡδ)overexpression on osteoclast differentiation and activity of downstream signaling molecules including c AMP-response element binding protein(CREB),extracellular regulated protein kinases(ERK),c-Jun N-terminal kinase(JNK)and p38 Mitogen-activated protein kinase(p38 MAPK)to elucidate the regulating of osteoclast differentiation by CaMKⅡδ.Methods 1 Screening of CaMKⅡδ transcript variant highly expressed during osteoclast differentiation and construction of recombinant vector for CaMKⅡδ overexpression.RAW264.7 cells were induced for osteoclast differentiate with 50ng/m L receptor activator of nuclear factor kappa B ligand(RANKL)and the cells were harvested at day 0,1,3 and 5 days after inuction.RT-PCR was used to identify the transcript variant of CaMKⅡδ highly expressed during osteoclast differentiation and the transcript variant.was used for construction of recombinant overexpression vector with lentivirus.RAW264.7 cells were transfected with recombinant vector and overexpression of CaMKⅡδ was verified by Real-time PCR and Western-blot.2 Effects of CaMKⅡδ overexpression on osteoclastogenesis,bone resorption and expression of downstream nuclear factor of activated T-cells c1(NFATc1)protein.Raw264.7 cells were divided into three groups: control group,blank vector group and CaMKⅡδ overexpression group.After virus thransfection,the cells were induced with 100ng/m L RANKL for osteoclast differentiation for 5 days and then the cells were harvested.Tartrate resistant acid phosphatase(TRAP)staining and dentin slices resorption lacunae analysis were used to identify osteoclast formation and bone resorption.Expression of NFATc1 protein in three groups was detected by immunofluorescent cytochemistry.3 Effects of CaMKⅡδ overexpression on activity of CREB,ERK,JNK and p38 proteins.RAW264.7 cells were divided into two groups: blank vector group and CaMKⅡδ overexpression group.The cells were induced with 100ng/m L RANKL for osteoclast differentiation.Western blotting was used to detect expression of total protein and phosphorylated protein of CREB,ERK,JNK and p38 at different time points.Results 1 RT-PCR examination showed that CaMKⅡδ transcript variant 2 and 3 highly expressed during osteoclast differentiation and recombinant overexpression lentivirus vector of CaMKⅡδ was successfully constructed with transcript variant 2. Overexpression of CaMKⅡδ was verified by Real-time PCR and Western-blot in RAW264.7 cells.Compared to control and blank vector group,m RNA level of CaMKⅡδ in overexpression group increased about 149.6%(P<0.05)and 95.3%(P<0.05)respectively,and protein level increased about 36.0%(P<0.05)and 32.7%(P<0.05)respectively.2 After induction with RANKL for 5 days,the number of TRAP-positive multinucleated osteoclasts,number and size of absorption lacunaes on dentin slices were 22.600±3.362,3.000±1.225 and 1429.980±759.394μm2 respectively in CaMKⅡδ overexpression group,which showed no significant differences with those in control group(23.600±3.050,2.600±1.517 and 1477.160±778.798μm2,respectively)and those in blank vector group(21.800±1.643,2.600±1.140 and 1322.720±225.484μm2,respectively)(P>0.05).Immunofluorescent cytochemistry showed that there were no obvious difference of fluorescent intensity for NFATc1 protein among three groups(P>0.05).These results suggested that CaMKⅡδ overexpression exhibied no significant effect on osteoclast formation,bone resorption and expression of NFATc1 protein.3 Overexpression of CaMKⅡδ showed no significant influence on protein activity of JNK and p38 during osteoclast differentiation(P>0.05).However,phosphorylation of ERK protein was significantly downregulated at 10 min,15min,30 min and 60 min under RANKL induction(P<0.05),and level of phosphorylated CREB was significantly upregulated at 0h and 1h(P<0.05).Conclusions 1 Recombinant lentivirus vector for CaMKⅡδ overexpression was successfully constructed with transcript variant 2 and RAW264.7 cell strain stably expressing recombinant CaMKⅡδ was established.2 CaMKⅡδ overexpression exhibited no significant effect on osteoclast formation,bone resorption,and expression of NFATc1 protein.3 Overexpression of CaMKⅡδ showed no significant influence on protein activity of JNK and p38 during osteoclast differentiation,but it could inhibit ERK phosphorylation,and up-regulat CREB protein phosphorylation.Above results suggest that although CaMKⅡδ overexpression,which is higher than normal level,could effect protein activity of some downstream signaling molecules,it is enough to exert significant influence on osteoclast differentiation.