10-kDa co-chaperonin regulates the expression levels of receptor activator of nuclear factor-κB ligand and osteoprotegerin in human osteoblasts.

The aim of the present study was to investigate the effect of recombinant (r-Mt) 10-kDa co-chaperonin (cpn10) on the expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) in third-generation cultured osteoblasts. The osteoblast-like cultures were isolated from bone fragments taken from patients undergoing surgery. Prior to stimulation with r-Mt cpn10, cells were incubated in serum-free medium for 24 h. r-Mt cpn10 was added into fresh serum-free medium, reaching final concentrations of 0.01-10 μg/ml. The levels of OPG were determined using enzyme-linked immunosorbent assay. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis was performed to determine the levels of RANKL and OPG mRNA. For measurement of the protein levels of OPG and RANKL, a western blotting assay was performed. r-Mt cpn10 downregulated the protein levels of OPG in the third generation cultured osteoblasts at a dose of 10 μg/ml. RT-qPCR revealed that the OPG mRNA level was decreased by 73% after 4 h and by 85.5% after 8 h following incubation with r-Mt cpn10 (10 μg/ml). Western blot analysis demonstrated similar results for the OPG protein level. In the third-generation cultured osteoblasts, the levels of RANKL mRNA and protein were increased by 2.6- and 1-fold, respectively, following incubation with r-Mt cpn10 (10 μg/ml). Furthermore, the RANKL/OPG ratio was markedly increased by r-Mt cpn10 (10 μg/ml) treatment. In conclusion, the results of the current study demonstrated that r-Mt cpn10 decreased the levels of OPG and increased the levels of RANKL in a dose- and time-dependent manner. Notably, the present study indicated that r-Mt cpn10 exerts its effect on osteoblastic cells by increasing the RANKL/OPG ratio.