ABSTRACT
Objective The aim of this study was to investigate the effect of Ca2+ /calmodulin - dependent kinase II (CaMKII)γ RNA interference on the expression of nuclear factor of activated T-cells cytoplasmic 1(NFATc1),tyrosine kinase(c-Src)and tartrate resistant acid phosphatase(TRAP)genes,and its role and molecular mechanism in osteoclast differentiation. Methods The CaMKII γ RNA interference vector was constructed by lentivirus and transfected into RAW264. 7 cells. The experiment was di-vided into three groups:A,B and C,which were the control group,negative vector group and interference vector group. After transfec-tion for 12 hours,osteoclasts induced by 50 ng/mL RANKL and the cells were harvested after induction for 5 days. Real-time quanti-tative PCR,Western blot and immunofluorescence were used to detect the expression of NFATc1,TRAP and c-Src genes in three groups. Results The mRNA levels of NFATc1,TRAP and c-Src in the group C decreased by 49. 86% ,43. 65% and 53. 57% ,re-spectively(P<0. 001),and the protein levels decreased by 54. 22% ,46. 75% and 45. 86% ,respectively(P<0. 001). There was no significant difference between the A and the B groups(P>0. 05). The fluorescence intensity of the above genes in the group C was significantly weaker than that in the A and B groups,and the formation of osteoclasts was significantly less than that in the A and B groups. Conclusion CaMKIIγ RNA interference significantly inhibited the expression of NFATc1,TRAP and c-Src genes,sugges-ting that CaMKIIγ plays a key regulatory role in osteoclast differentiation.
ABSTRACT
Objective To investigate the function of cAMP response element binding protein (CREB) and extracellular signal-regulated kinase (ERK1/2) in osteoclast differentiation mediated by Ca2+/calmodulin-dependent kinase Ⅱ (CaMK Ⅱ)δ,and elucidate the molecular mechanism thereof.Methods CaMK Ⅱδ RNA interference lentivirus vector was constructed and mouse RAW264.7 cells were transfected with the virus to determine the interference efficiency.After virus transfection,RAW264.7 cells were treated with 50ng/ml receptor activator of nuclear factor κB ligand (RANKL) and the phosphorylation levels of CREB and ERK1/2 were detected at different time points.The cells were also treated with PD98059,an ERK1/2 inhibitor,to determine the effect of ERK1/2 signal blocking on the expression of nuclear factor activated T-cells cytoplasmic 1 (NFATc1) and osteoclast differentiation.Results Interference efficiency of recombinant CaMK Ⅱδ virus vector was 77.2％ at mRNA level and 70.2％ at protein level.CaMK Ⅱδ RNA interference significantly suppressed phosphorylation of CREB and ERK1/2,and the levels ofp-CREB and p-ERK1/2 were down-regulated by 21％-55％ and 55％-64％,respectively.ERK1/2 inhibitor significantly down-regulated the protein expression of NFATc1,and the number of osteoclast,the number and size of bone resorption lacunae decreased by 39.3％,50.0％ and 52.3％,respectively.Conclusion CaMK Ⅱδ RNA interference may significantly suppress the phosphorylation of CREB and ERK1/2,and CREB and ERK1/2 have mediated the CaMK Ⅱδ-induced osteoclast differentiation.
ABSTRACT
Objective To study the effect of zoledronate (ZOL) on Ca2+/calmodulin-dependent kinase Ⅱ δ (CaMK Ⅱ δ) and down-stream gene expressions during osteoclast differentiation.Methods Mouse osteoclast precursors RAW264.7 cells were divided into the control group and ZOL group.The cells in both groups were induced with 50μg/L receptor activator of nuclear factor kappa B ligand (RANKL) and were harvested on 5 d,while the cells in ZOL group were also simultaneously treated with 1 × 10-6 mol/L ZOL for 2 d.Five days later,the cells were harvested and examined osteoclastogenesis,as well as gene expressions of CaMK Ⅱ δ,nuclear factor of activated T-cells cytoplasmic 1 (NFATc1),tartrate-resistant acid phosphatase (TRAP) and cell-sarcoma receptor coactivator (c-Src).Results The number of TRAP positive multinuclear osteoclasts,number and size of dentin absorption lacunae and area in the ZOL group were (20.0±3.2),(18.0±4.2) and (6 335.3± 1 043.2)μm2 respectively,which were significantly lower than (36.0 ± 8.4),(37.2 ± 5.0) and (11 636.2 ± 3 661.1) μm2 in the control group and decreased by 44.4 ％,51.6 ％ and 45.6 ％ respectively (P＜0.01).ZOL also significantly inhibited the gene expressions of CaMK Ⅱ δ,NFATc1,TRAP and c-Src,and the mRNA levels of these genes were decreased by 44.1％,49.0％,53.8％ and 49.6％ respectively,the protein level were decreased by 43.5 ％,32.2 ％,45.5 ％ and 48.0 ％ respectively.The immunofluorescent cytochemistry detection results showed the fluorescence intensity of CaMK Ⅱ δ,NFATc1,TRAP and c-Srcin in the ZOL group was significantly weakened when compared with the control group.Conclusion ZOL could significantly inhibit the osteoclast formation and bone absorption function,and down-regulates gene expressions of CaMK Ⅱ δ,NFATc1,TRAP and c-Src in osteoclast differentiation.