FTY-720P Suppresses Osteoclast Formation by Regulating Expression of Interleukin-6 (IL-6), Interleukin-4 (IL-4), and Matrix Metalloproteinase 2 (MMP-2)
Dawei Zhang, Yongjun Huang, Zongwen Huang, Rongkai Zhang, Honggang Wang, Dong Huang
(Section 2, Department of Orthopedics, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, Guangdong, China (mainland))
Med Sci Monit 2016; 22:2187-2194
Osteoclast formation is closely related to the immune system. FTY720, a new immunosuppressive agent, has some functions in immune regulation. Its main active ingredients become FTY-720P in vivo by phosphorylation modification. The objective of this study was to determine the effects of FTY-720 with various concentrations on osteoclasts in vitro.
MATERIAL AND METHODS: RAW264.7 cells and bone marrow-derived mononuclear phagocytes (BMMs) were treated with RANKL to obtain osteoclasts in vitro. To investigate the role of FTY-720 in osteoclast formation, trap enzyme staining was performed and the number of osteoclasts was counted. Bone slices were stained with methylene blue, we counted the number of lacunae after bone slices were placed into dishes together with osteoclasts, and we observed the effect and function of FTY-720 in osteoclasts induced by RAW264.7 cells and BMMs. Then, we used a protein array kit to explore the effects of FTY-720P on osteoclasts.
RESULTS: The results of enzyme trap staining and F-actin staining experiments show that, with the increasing concentration of FTY-720P, the number of osteoclast induced by RAW264.7 cells and BMMs gradually decreased (P<0.05), especially when the FTY-720P concentration reached 1000 ng/ml, and the number of osteoclasts formed was the lowest (P<0.05). With bone lacuna toluidine blue staining, the results also show that, with the increasing concentration of FTY-720P, the number of bone lacuna gradually decreased (P<0.05), and the number of lacunae is lowest when the concentration reached 800 ng/ml. Finally, protein array results showed that IL-4, IL-6, IL-12, MMP-2, VEGF-C, GFR, basic FGF, MIP-2, and insulin proteins were regulated after FTY-720P treatment.
CONCLUSIONS: FTY-720P can suppress osteoclast formation and function, and FTY-720P induces a series of cytokine changes.
Keywords: Interleukin-4 - biosynthesis, Cell Line, Bone Marrow Cells - metabolism, Animals, Interleukin-6 - biosynthesis, Macrophages - metabolism, Male, Matrix Metalloproteinase 2 - biosynthesis, Mice, Organophosphates - pharmacology, Osteoclasts - metabolism, RANK Ligand - pharmacology, RAW 264.7 Cells, Sphingosine - pharmacology, Vascular Endothelial Growth Factor C - metabolism