[Tumor necrosis factor-α promotes osteoclast differentiation via sialylation in mice]

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.

Keywords: osteoclasts; sialylation; tumor necrosis factor-α.

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野生型小鼠及TNF-α转基因小鼠膝关节软骨下骨TRAP染色(红） Subchondral TRAP staining (red) of the knee joint in wild-type mice and TNF-αtransgenic mice.

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野生型小鼠及TNF-α转基因小鼠膝关节软骨下骨结构（骨量）变化 Changes in subchondral bone structure (bone mass) of the knee joint in wild-type mice and TNF-αtransgenic mice.

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野生型小鼠及TNF-α转基因小鼠膝关节软骨下骨TRAP（红）及唾液酸（绿）免疫荧光共定位染色 TRAP (red) and SA (green) co-staining in subchondral bone of the knee joint in wild-type mice and TNF-α transgenic mice.

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两组破骨细胞前体细胞系TRAP（红）及唾液酸（绿）表达 TRAP (red) and SA (green) expression in osteoclast precursor cells of the two groups.

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不同干预下破骨细胞前体细胞系的TRAP(红）及其唾液酸（绿）的表达情况 TRAP (red) and sialic acid (green) expressions of osteoclast precursor cells with different interventions.

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不同干预下破骨细胞前体细胞系TRAP的表达情况 Expression of TRAP in osteoclast precursor cells with different interventions.

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不同干预下破骨细胞前体细胞系TRAP mRNA转录水平 TRAP mRNA levels in osteoclast precursor cells with different interventions.

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各组原代破骨细胞TRAP表达情况 TRAP expression in primary osteoclasts in each group.

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各组原代破骨细胞唾液酸（绿）表达情况 Sialic acid (green) expression in primary osteoclasts in each group.