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Effect of zoledronate on protein interaction between Ca2+/calmodulin-dependent protein kinase Ⅱ and calmodulin and expression of downstream genes during osteoclast differentiation

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Author:
No author available
Journal Title:
Chinese Journal of Stomatology
Issue:
2
DOI:
10.3760/cma.j.issn.1002-0098.2017.02.014
Key Word:
钙-钙调蛋白依赖性蛋白激酶2;钙调蛋白;唑来膦酸;免疫沉淀法;Calcium-calmodulin-dependent protein kinase type 2;Calmodulin;Zolderonate;Immunoprecipitation

Abstract´╝Ü Objective To investigate the effect of zoledronate on protein interaction between Ca2+/ calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) and calmodulin and protein expression of nuclear factor of activation of T cells-1 (NFATc1) and tartrate resistant acid phosphatase (TRAP) during osteoclast differentiation.Methods Mouse RAW264.7 cells were divided into group A and B and were cultured.Group A was induced with 50 mg/L receptor activator of NF-κB ligand (RANKL) for osteoclastogenesis,and group B was treated with 1 × l0-6 zoledronate for two days from day 2.Co-immunoprcipitation (Co-IP) and reverse Co-IP were used to detect the protein-binding between CaMK Ⅱ and calmodulin.Western-blotting and immunofluorescent cytochemistry were also used to detect the protein level of NFATc 1 and TRAP in both groups.Osteoclast formation was also analyzed.Results In group B,the number of osteoclasts,number and size of dentin resorption lacunaes were 11.3±1.5,8.7±2.1 and (5 034.4±775.4) μm2respevtively,which were significantly lower than those (37.7±5.7,23.0±4.0 and [15 042.7± 1 906.0] μm2) in group A (P<0.01).Co-IP and reverse Co-IP examination indicated that protein-binding between CaMK Ⅱ and calmodulin significantly decreased by 59.8% and 50.9% in group B compared with group A (P<0.01).The protein level of calmodulin and CaMK lⅡ in total cellular proteins also significantly decreased by 52.1% and 51.5% in group B compared with group A (P<0.01).NFATc1 and TRAP protein decreased by 52.4% and 38.9% in group B than in group A (P<0.01),respectively.Conclusions Zoledronate could significantly inhibit protein-binding between CaMK Ⅱ and calmodulin and down-regulate protein level of NFATc1 and TRAP.

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