Background
α-Lipoic acid (α-LA), a thiol antioxidant, suppressed TNF-α-induced apoptosis of human bone marrow stromal cells (hBMSC) in our previous study, suggesting that α-LA may stimulate bone formation. In the present study, we examined effects of α-LA on protein secretions into hBMSC culture media in order to investigate whether α-LA can modulate cell-to-cell interactions, and noted that α-LA stimulated secreted receptor activator of NF-κB ligand (RANKL). Therefore, we examined the effect of α-LA on osteoclastogenesis and bone resorption.
Method
After incubation of HS-5 cells with or without α-LA, we performed 2-dimensional electrophoresis and MALDI-TOF analysis. RANKL and osteoprotegerin levels were determined by ELISA assay. Tartrate-resistant acid phosphatase (TRAP) staining was performed in order to evaluate osteoclastogenesis, both in a coculture system of mouse bone marrow cells and osteoblasts, and in a mouse bone marrow cell culture system. Bone resorption was evaluated using dentine slices.
Results
Using proteomic analysis, we identified six differentially expressed proteins in the conditioned media of α-LA-treated hBMSC. One of these proteins, RANKL was significantly up regulated, as confirmed by immunoblotting with anti-RANKL antibody. ELISA assay showed that α-LA stimulated RANKL production in cellular extracts (membranous RANKL) about 5-fold and in conditioned media (soluble RANKL) about 23-fold, but had no effect on osteoprotegerin secretion. Despite increasing the RANKL/OPG ratio, α-LA showed a dose-dependent suppression of osteoclastogenesis, both in a coculture system of mouse bone marrow cells and osteoblasts, and in a mouse bone marrow cell culture system, and reduced bone resorption. In addition, α-LA-induced soluble RANKL was not inhibited by matrix metalloprotease inhibitors, indicating that soluble RANKL is produced by α-LA without any posttranslational processing.
Conclusion
These results suggest that α-LA suppresse
