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Abstract
The aim of the present study was to investigate the effect of recombinant Mycobacterium tuberculosis (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.
Highlights
Osteoblasts and osteoclasts maintain normal bone metabolism balance, and are involved in bone metabolism and the immune activity of the body [1]
To assess the effect of recombinant Mycobacterium tuberculosis (r‐Mt) cpn10 on OPG protein secretion, the third‐generation cultured osteoblasts were treated with r‐Mt cpn10 at doses ranging from 0.01 to 10 μg/ml in serum‐free media
The results revealed that lower doses (0.01, 0.1 and 1 μg/ml) of r‐Mt cpn10 failed to significantly decrease OPG protein secretion; 10 μg/ml r‐Mt cpn10 significantly decreased OPG protein secretion by 50% compared with the control (P
Summary
Osteoblasts and osteoclasts maintain normal bone metabolism balance, and are involved in bone metabolism and the immune activity of the body [1]. Receptor activator of nuclear factor‐κB ligand (RANKL) expression, which can be found on the surface of osteoblasts, is activated by stimulation with endocrine hormones and local cytokines, and can be combined with RANK receptors on the surface of osteoclasts to induce osteoclast proliferation and activation [2]. Osteoblasts and bone marrow stromal cells express RANKL to promote osteoclast differentiation, enhance the viability of mature osteoclasts and prevent osteoclast apoptosis. Certain cells, including bone marrow stromal cells and osteoblasts, express OPG, which inhibits the differentiation and maturation of osteoclasts to decrease bone resorption activity and induce apoptosis. Regulators, including serum osteoprotegerin, serve a function in the homeostasis of bone resorption and reconstruction, and the whole process is regulated by osteoblasts [6,7]
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