Effects of Immunosuppressants on RANKL-induced Osteoclast Differentiation of RAW264.7 Cells

Objectives: Some therapeutic strategies to treat bone diseases such as osteoporosis aimed at decreasing osteoclast activity. Our previous study demonstrated that ascomycin inhibited the number of round shaped-osteoclast cells (ROC) and the mRNA expression of osteoclast specific genes in RAW264.7 cells. The present study was designed to examine effect of immunosuppressant cyclosporine A, rapamycin, and tacrolimus on the formation and activity of osteoclasts on RANKL-induced RAW264.7 cells.
Methods: The effects of immunosuppressant on osteoclasts were examined by measuring the following: (1) the cell viability, (2) the formation of tartrate-resistant acid phosphatase (TRAP) + multinucleated cells, (3) ROC formation, and (4) the mRNA expression of osteoclast-associated genes.
Results: Cyclosporine A inhibited RANKL-stimulated TRAP activity and ROC formation of RAW264.7 cells in a dose-dependent manner (10 nM, 100 nM, and 1 uM). Tacrolimus also significantly decreased TRAP activity in the concentration of 20 μM and inhibited ROC formation in 0.1 nM. In addition, the RANKL-stimulated induction of NFATc1 transcription factors was abrogated by cyclosporine A and tacrolimus. In contrast, rapamycin induced TRAP activity and ROC formation in concentration of 100 nM, and increased NFATc1 gene expression by 12-fold compared to RANKL-treated cells.
Conclusions: Taken together, cyclosporine A and tacrolimus was shown to inhibit RANKL-induced osteoclast differentiation of RAW264.7 cells, suggesting that therapeutic effects on bone-destructive processes. On the other hand, rapamycin enhances osteoclastogenesis and may affect bone metabolism in vivo after long-term use.