Expression and function of transmembrane-4 superfamily (tetraspanin) proteins in osteoclasts: reciprocal roles of Tspan-5 and NET-6 during osteoclastogenesis.
Expression and function of transmembrane-4 superfamily (tetraspanin) proteins in osteoclasts: reciprocal roles of Tspan-5 and NET-6 during osteoclastogenesis.
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DOI：
10.2332/allergolint.o-07-488
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发表时间：
2007-12-01
期刊：
影响因子：
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通讯作者：
Saeki, Yukihiko
中科院分区：
文献类型：
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作者：
Iwai, Kaori;Ishii, Masaru;Saeki, Yukihiko
背景技术背景：破骨细胞是骨重建所必需的骨吸收多核多核体，在RANK/RANKL系统刺激下通过单核巨噬细胞/单核细胞谱系前体细胞的细胞融合形成。最近的研究表明，四跨膜蛋白家族，如CD 9，在这些细胞类型的细胞融合/多核体形成中起关键作用。然而，到目前为止，有有限的知识类型的tetraspanins中表达的破骨细胞和他们的precurs.METHODS：不同的tetraspanin蛋白的表达在单核/巨噬细胞系破骨细胞前体细胞系，RAW 264.7，cyclopedically研究使用特异性引物的RT-PCR和定量实时RT-PCR。两种四跨膜蛋白Tspan-5和NET-6的功能，其表达模式被RANKL刺激改变，通过将基因特异性短干扰RNA导入这些细胞类型来检测。在哺乳动物造血细胞中的17种四跨膜蛋白中，RAW264.7细胞表达12种不同类型的四跨膜蛋白的mRNA，即，CD 9、CD 37、CD 53、CD 63、CD 81、CD 82、CD151、NAG-2、NET-6、SAS、Tspan-3和Tspan-5。有趣的是，在RANKL刺激后，它们成熟为破骨细胞期间，Tspan-5的转录物上调，而NET-6的转录物下调。通过基因特异性RNA干扰靶向抑制Tspan-5抑制破骨细胞生成过程中RANKL诱导的细胞融合，而抑制NET-6增强破骨细胞生成本身。这些结果表明，Tspan-5和NET-6在破骨细胞生成过程中具有相互作用，即，Tspan-5和NET-6分别进行正性和负性调节。RANKL调节破骨细胞生成通过改变这些四跨膜蛋白的平衡。结论：这些数据表明，多样性的四跨膜蛋白表达在破骨细胞的前体细胞，并在破骨细胞生成过程中的细胞融合是由不同的四跨膜蛋白家族蛋白，如Tspan-5和NET-6的合作。这项研究表明，四跨膜蛋白家族蛋白的功能改变可能具有治疗潜力的疾病，其中破骨细胞发挥了重要作用，如类风湿性关节炎和骨质疏松症。
BACKGROUND: Osteoclasts are bone-resorbing multinuclear polykaryons essential for bone remodeling, formed through cell fusion of mononuclear macrophage/monocyte lineage precursor cells upon stimulation by the RANK/RANKL system. Recent studies have revealed that a family of tetraspanin proteins, such as CD9, is critically involved in the cell fusion/polykaryon formation of these cell types. Until now, however, there is limited knowledge about the types of tetraspanins expressed in osteoclasts and their precursors.METHODS: The expression of different tetraspanin proteins in a monocyte/macrophage-lineage osteoclast precursor cell line, RAW264.7, was cyclopedically investigated using RT-PCR with specific primers and quantitative real-time RT-PCR. The function of two kinds of tetraspanins, Tspan-5 and NET-6, whose expression pattern was altered by RANKL stimulation, was examined by transfecting gene-specific short-interfering RNAs into these cell types.RESULTS: Of the 17 tetraspanins in mammalian hematopoietic cells, RAW264.7 cells express mRNA for 12 different kinds of tetraspanins, namely, CD9, CD37, CD53, CD63, CD81, CD82, CD151, NAG-2, NET-6, SAS, Tspan-3, and Tspan-5. Interestingly, during their maturation into osteoclasts upon RANKL stimulation, the transcript for Tspan-5 is up-regulated, whereas that for NET-6 is down-regulated. Targeted inhibition of Tspan-5 by using gene-specific RNA interference suppressed RANKL-induced cell fusion during osteoclastogenesis, whereas inhibition of NET-6 augmented the osteoclastogenesis itself. These results suggest that Tspan-5 and NET-6 have a reciprocal function during osteoclastogenesis, i.e., positive and negative regulation by Tspan-5 and NET-6, respectively. RANKL regulates osteoclastogenesis by altering the balances of these tetraspanin proteins.CONCLUSIONS: These data indicate that a diversity of tetraspanins is expressed in osteoclast precursors, and that cell fusion during osteoclastogenesis is regulated by cooperation of distinct tetraspanin family proteins such as Tspan-5 and NET-6. This study indicates that functional alterations of tetraspanin family proteins may have therapeutic potential in diseases where osteoclasts play a major role, such as rheumatoid arthritis and osteoporosis.