Interaction between S100A8/A9 and Annexin A6 Is Involved in the Calcium-induced Cell Surface Exposition of S100A8/A9
Interaction between S100A8/A9 and Annexin A6 Is Involved in the Calcium-induced Cell Surface Exposition of S100A8/A9
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DOI：
10.1074/jbc.m803908200
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发表时间：
2008-11-14
影响因子：
4.8
通讯作者：
Nacken, Wolfgang
中科院分区：
文献类型：
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作者：
Bode, Guenther;Lueken, Aloys;Nacken, Wolfgang
钙结合 S100A8/A9 复合物（MRP8/14；钙颗粒蛋白）被认为是急性和慢性炎症中重要的促炎介质，并且最近作为在各种人类癌症中上调的分子标记而受到关注。在此，我们报道 S100A8/A9 在乳腺癌细胞系中表达，并在 SKBR3 和 MCF-7 细胞中被白细胞介素 1 β 和肿瘤坏死因子 α 上调。我们通过亲和层析将磷脂结合蛋白膜联蛋白 A6 鉴定为潜在的 S100A8/A9 结合蛋白。这一发现通过 Southwestern 重叠实验和 S100A8/A9 特异性单克隆抗体 27E10 的免疫共沉淀得到了验证。免疫细胞化学实验表明，S100A8/A9 和膜联蛋白 A6 在 SKBR3 乳腺癌细胞中主要共定位于膜结构。钙流入后，S100A8/A9 和膜联蛋白 A6 都暴露在 SKBR3 细胞的细胞表面。亚细胞分离研究表明，A23187 刺激后，S100A8/A9 的膜结合并未增强。然而，钙流入后，S100A8/A9 和膜联蛋白 A6 都暴露在 SKBR3 细胞的细胞表面。人工脂质体实验表明，S100A8/A9 能够独立于膜联蛋白 A6 和钙而与膜结合。最后，在 A23187 处理的 A431 和 HaCaT 细胞中无法观察到 S100A8/A9 的细胞表面表达。已知这两种细胞系均不含膜联蛋白 A6。在 SKBR3 细胞中通过小干扰 RNA 抑制膜联蛋白 A6 的表达会消除钙流入时 S100A8/A9 的细胞表面暴露，表明膜联蛋白 A6 有助于膜相关的 S100A8/A9 复合物的钙依赖性细胞表面暴露。
The calcium binding S100A8/A9 complex (MRP8/14; calgranulin) is considered as an important proinflammatory mediator in acute and chronic inflammation and has recently gained attention as a molecular marker up-regulated in various human cancers. Here, we report that S100A8/A9 is expressed in breast cancer cell lines and is up-regulated by interleukin-1 beta and tumor necrosis factor-alpha in SKBR3 and MCF-7 cells. We identified the phospholipid-binding protein annexin A6 as a potential S100A8/A9 binding protein by affinity chromatography. This finding was verified by Southwestern overlay experiments and by coimmunoprecipitation with the S100A8/A9-specific monoclonal antibody 27E10. Immunocytochemical experiments demonstrated that S100A8/A9 and annexin A6 colocalize in SKBR3 breast cancer cells predominantly in membranous structures. Upon calcium influx both S100A8/A9 and annexin A6 are exposed on the cell surface of SKBR3 cells. Subcellular fractionation studies suggested that after A23187 stimulation membrane association of S100A8/A9 is not enhanced. However, both S100A8/A9 and annexin A6 are exposed on the cell surface of SKBR3 cells upon calcium influx. Experiments with artificial liposomes indicated that S100A8/A9 is able to associate with membranes independently of both annexin A6 and independently of calcium. Finally, cell surface expression of S100A8/A9 could not be observed in A23187-treated A431 and HaCaT cells. Both cell lines are known to be devoid of annexin A6. Repression of annexin A6 expression by small interfering RNA in SKBR3 cells abolishes the cell surface exposition of S100A8/A9 upon calcium influx, suggesting that annexin A6 contributes to the calcium-dependent cell surface exposition of the membrane associated-S100A8/A9 complex.