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

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.