| Objectives Ligand of receptor activator of nuclear factor ?B(RANKL) was used in this study to induce RAW264.7 cells, a mice leukemia mononuclear macrophage cell line, to differentiation into osteoclasts. During the process, zoledronate, a third generation of biphosphonates, was added into cells and its inhibition effect on osteoclastogenesis and expression of nuclear factor of activated T cells type c1(NFATc1), a master regulator of osteoclasto-specific genes, as well as other downstream molecules was detected. Recombinant mouse NFATc1 and recombinant constructive active human NFATc1 were constructed with a lentivirus vector to verify rescue effect of NFATc1 over-expression on zoledronate-induced inhibition of osteoclastogenesis.Methods 1 RAW264.7 cells was cultured in vitro and the cells were divided into two groups, group A and B. Both groups were induced with 50ng/ml RANKL for 5 days for osteoclastogenesis, while cells in group B were also treated with 1 × 10-6mmol/ml zoledonate from the 2nd day for 2 days. At day 6, the cells were harvested and tartrate resistant acid phosphatase(TRAP) staining, as well as dentin slice resorption analysis was applied for detection of osteoclastogenesis and bone resorption function. Gene expression of NFATc1, TRAP and c-Src were also detected by Western-Blot, Real-time PCR and immunofluorescent cytochemistry, respectively. 2 Recombinant mouse NFATc1 and recombinant human constructive active NFATc1(ca-NFATc1) were constructed and a blank lentivirus vector with green fluorescence was used to transfect RAW264.7 cells to determine the most suitable MOI value for transfection. Then RAW264.7 cells were transfected with recombinant NFATc1 and gene expression of NFATc1, at both protein and m RNA level, was deteted by Western-Blot and Real-time, respectively, for verification of overexpression of NFATc1. 3 RAW264.7 cells were divided into 3 groups: group A served as control, group B was transfected with negative vector, and group C was transfected with recombinant NFATc1. The cells in all three groups were treated with RANKL and zoledronate with the same method mentioned above. Five days later, the cells were harvested and many examinations, including TRAP staining, dentin slices resorption analysis, Western-Blot, Real-time PCR and immunofluorescent cytochemistry,were performed to verify rescue effect of recombinant NFATc1 on zoledronate-induced inhibition of osteoclastogenes and expression of osteoclast-specific genes.Results 1 After treatment with RANKL and zoledronate, multinuclear osteoclasts were observed in both group A and B. The number of osteoclasts, number and size of absorption lacunaes on dentin slices in group B were 24.50±2.10, 22.30±3.10, 5746.00±786.60, respectively, which were all significantly lower than those in group A(P<0.05), which were 55.00±3.00, 32.70±2.50, 9054.70±182.50, respectively. Western-Blot showed that significant decrease of protein level of NFATc1, TRAP and c-Src in group B when compared with group A and the decreases were 44.10%, 37.90% and 35.80%, respectively(P<0.05). m RNA level of above three genes in group B also decreased 50.50%, 49.50% and 45.50% respectively when compared with A demonstrated by Real-time PCR(P<0.05).Smilar results were also achieved by immunofluorescent cytochemistry which showed a significant decrease of protein level of above tree genes in group B when compared with group A. Above results indicated that zoledronate inhibited osteoclastogenesis from RAW264.7 cells and downregulated gene expression of osteoclast-related genes including NFATc1, TRAP and c-Src. 2 Successful construction of recombinant mouse and human ca-NFATc1 were confirmed by NFATc1 expression in RAW264.7 cells. The most suitable MOI value for transfection was 30. Under such condition, high transfection rate, good cell viability and proliferation capacity could be achieved. Transfection with recombinant mouse NFATc1 in group C led to a significant increase of protein level of NFTAc1 when compared with group A and the increase were 178.60%?127.80%(P<0.01). m RNA level of NFTAc1 in group C also increased about 41.90% ?33.60%(P<0.05) than that in group A. Whereas both protein level and m RNA level of NFATc1 and TRAP in group A were similar to those in group B(P>0.05). Similar results were obtained when recombinant human ca-NFATc1 were used for cell transfection. 3 Although all three groups were treated with both RANKL and zoledronate, TRAP positive multinuclear osteoclasts, the number and size of absorption lacunaes on dentin slices in group C were 71.00±4.60, 42.30±1.50 and 11863.70±968.10mm2 respectively, which were all significantly higher than those(27.0±1.00,23.00±1.70 and 6024.00±270.80mm2) in group A(P<0.01).Cells in group C transfected with recombinant mouse NFATc1 showed significant increase of protein level of NFTAc1, TRAP and c-Src when compared with group A and the increase were 92.00%, 90.20% and 122.00% respectively(P<0.01). m RNA level of above three genes in group C also increased about 70.20%, 71.70% and 69.20% compared with group A(P<0.01). Whereas all above parameters in group B were similar to those in group A(P>0.05). Similar results were obtained when cells in group C were transfected with recombinant human ca-NFATc1. Above results suggested that overexpression of exogenic recombinant NFATc1 could rescue the inhibition effect of zoledronate on osteoclastogenesis and expression of osteoclast-related genes including NFATc1, TRAP and c-Src.Conclusions Zoleronate acid could inhibit osteoclast formation from RAW264.7 cells and expression of osteoclast-related genes including NFATc1, TRAP and cSrc.Constructed the recombinant NFATc1 succefully and promote the expression of TRAP.Over-expression of NFATc1 could rescue above inhibition effect of zoledronic on osteoclastogenesis. NFATc1, as a key mediator for Ca2+ signal axis and master regulator of osteoclast-related genes, may play a critical role in zoledronate-induced inhibition of osteoclastogenesis. |
PDF Full Text Request