Takahata Masahiko, Iwasaki Norimasa, Nakagawa Hiroaki, Abe Yuichiro, Watanabe Takuya, Ito Manabu, Majima Tokifumi, Minami Akio
Department of Orthopaedic Surgery, Hokkaido University Graduate School of Medicine, Kita-15 Nishi-7, Kita-ku, Sapporo 060-8638, Japan.
Bone. 2007 Jul;41(1):77-86. doi: 10.1016/j.bone.2007.03.016. Epub 2007 Apr 5.
Sialic acid, which is located at the end of the carbohydrate moiety of cell surface glycoconjugates, is involved in many biologic responses, such as intercellular reactions and virus-cell fusion, especially in hematopoietic cells. Here we provide experimental evidence that the sialic acid of cell surface glycoconjugates has a role in osteoclast differentiation. Lectin histochemical study demonstrated the existence of both alpha (2,3)-linked-sialic acid and alpha (2,6)-linked-sialic acid in mouse bone marrow-derived macrophages and in the RAW264.7 macrophage cell line, which are osteoclast precursors. Flow cytometric analysis of surface lectin staining revealed the kinetics of these sialic acids during osteoclastogenesis: alpha (2,3)-linked-sialic acid was abundantly expressed throughout osteoclastogenesis, whereas alpha (2,6)-linked-sialic acid levels declined at the terminal stage of osteoclast differentiation. To investigate the role of sialic acid in osteoclast differentiation, we performed an osteoclastogenesis assay with or without exogenous sialidase treatment. Desialylated cells formed TRAP-positive mononuclear cells, but did not become multinuclear cells despite the normal expression of osteoclast markers such as cathepsin K, integrin beta3, and nuclear factor-ATc1. Flow cytometric analysis also demonstrated that exogenous sialidase effectively removed alpha (2,6)-linked-sialic acid, but only slightly changed the alpha (2,3)-linked-sialic acid content, suggesting that alpha (2,6)-linked-sialic acid might be involved in osteoclast differentiation. Findings from knockdown analysis using small interfering RNA oligonucleotides against alpha 2,6-sialyltransferase support this idea: alpha (2,6)-linked-sialic acid-deficient cells markedly inhibit the formation of multinuclear osteoclasts. Our findings suggest that alpha (2,6)-linked-sialic acid of cell surface glycoconjugates has a role in osteoclast differentiation, possibly via its role in the cell-cell fusion process.
唾液酸位于细胞表面糖缀合物碳水化合物部分的末端，参与许多生物学反应，如细胞间反应和病毒-细胞融合，尤其在造血细胞中。在此，我们提供实验证据表明细胞表面糖缀合物的唾液酸在破骨细胞分化中起作用。凝集素组织化学研究表明，在破骨细胞前体——小鼠骨髓来源的巨噬细胞和RAW264.7巨噬细胞系中，同时存在α(2,3)-连接的唾液酸和α(2,6)-连接的唾液酸。表面凝集素染色的流式细胞术分析揭示了破骨细胞生成过程中这些唾液酸的变化动态：α(2,3)-连接的唾液酸在整个破骨细胞生成过程中大量表达，而α(2,6)-连接的唾液酸水平在破骨细胞分化末期下降。为了研究唾液酸在破骨细胞分化中的作用，我们进行了有或无外源性唾液酸酶处理的破骨细胞生成试验。去唾液酸化的细胞形成了抗酒石酸酸性磷酸酶（TRAP）阳性的单核细胞，但尽管破骨细胞标志物如组织蛋白酶K、整合素β3和活化T细胞核因子c1（nuclear factor-ATc1）正常表达，却未变成多核细胞。流式细胞术分析还表明，外源性唾液酸酶有效地去除了α(2,6)-连接的唾液酸，但仅轻微改变了α(2,3)-连接的唾液酸含量，这表明α(2,6)-连接的唾液酸可能参与破骨细胞分化。使用针对α2,6-唾液酸转移酶的小干扰RNA寡核苷酸进行的敲低分析结果支持了这一观点：缺乏α(2,6)-连接唾液酸的细胞显著抑制多核破骨细胞的形成。我们的研究结果表明，细胞表面糖缀合物的α(2,6)-连接唾液酸在破骨细胞分化中起作用，可能是通过其在细胞-细胞融合过程中的作用。
