Abstract: Objective To explore the mechanism through which tumor necrosis factor-α (TNF-α) promotes osteoclast differentiation. Methods Bilateral knee joint samples were collected from 4-month-old wild-type mice and TNF-α transgenic mice for CT scan analysis, TRAP staining and sialic acid staining analysis. The osteoclast precursor (RAW264.7) cells were cultured for 3 days in induction medium in the presence of vehicle, TNF-α, or TNF-α and sialidase, and were then examined with RT-qPCR, TRAP staining, and sialic acid immunofluorescence co-localization staining. Bone marrow-derived macrophages isolated from the wild-type mice and TNF-α transgenic mouse and cultured in induction medium with or without the addition of sialidase, and TRAP and sialic acid staining was performed after 3 days of cell culture. Results TRAP staining showed that the number of osteoclasts increased significantly in TNF-α transgenic mice as compared with the wild-type mice (P<0.0001), and micro-CT analysis revealed significant reductions of BV/TV, Tb.N, and Tb.Th in TNF-α transgenic mice (P<0.001). The osteoclasts in TNF-α transgenic mice also showed a significantly increased expression of sialic acid (P=0.004). In the cell experiment, RAW264.7 cells cultured with TNF-α showed a significantly higher expression of sialic acid (P<0.0001) and a greater osteoclast formation rate (P=0.0007) than the the control cells, while the addition of sialidase significantly reduced sialic acid expression, osteoclast formation rate and TRAP mRNA level in TNF-α-treated cells (P<0.0001). Similarly, in the bone marrow-derived macrophages, sialic acid expression and osteoclast formation rate were significantly increased by incubation with TNF-α (P<0.0001), but the increments were obviously reduced by addition of sialidase in the medium (P<0.0001). Conclusion TNF-α can promote the differentiation and activity of osteoclasts by increasing the sialylation level in the osteoclasts.

Key words: tumor necrosis factor-α; sialylation; osteoclasts