1] Department of Hematology, Chiba University Hospital, Chiba, Japan [2] Department of Clinical Cell Biology and Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.
1] Department of Hematology, Chiba University Hospital, Chiba, Japan [2] Department of Clinical Cell Biology and Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan [3] Division of Transfusion Medicine and Cell Therapy, Chiba University Hospital, Chiba, Japan.
Exp Mol Med. 2014 Apr 4;46(4):e89. doi: 10.1038/emm.2013.161.
LR11, also known as SorLA or SORL1, is a type-I membrane protein from which a large extracellular part, soluble LR11 (sLR11), is released by proteolytic shedding on cleavage with a disintegrin and metalloproteinase 17 (ADAM17). A shedding mechanism is presumed to have a key role in the functions of LR11, but the evidence for this has not yet been demonstrated. Tetraspanin CD9 has been recently shown to regulate the ADAM17-mediated shedding of tumor necrosis factor-α and intercellular adhesion molecule-1 on the cell surface. Here, we investigated the role of CD9 on the shedding of LR11 in leukocytes. LR11 was not expressed in THP-1 monocytes, but it was expressed and released in phorbol 12-myristate 13-acetate (PMA)-induced THP-1 macrophages (PMA/THP-1). Confocal microscopy showed colocalization of LR11 and CD9 proteins on the cell surface of PMA/THP-1. Ectopic neo-expression of CD9 in CCRF-SB cells, which are LR11-positive and CD9-negative, reduced the amount of sLR11 released from the cells. In contrast, incubation of LR11-transfected THP-1 cells with neutralizing anti-CD9 monoclonal antibodies increased the amount of sLR11 released from the cells. Likewise, the PMA-stimulated release of sLR11 increased in THP-1 cells transfected with CD9-targeted shRNAs, which was negated by treatment with the metalloproteinase inhibitor GM6001. These results suggest that the tetraspanin CD9 modulates the ADAM17-mediated shedding of LR11 in various leukemia cell lines and that the association between LR11 and CD9 on the cell surface has an important role in the ADAM17-mediated shedding mechanism.
LR11，也称为 SorLA 或 SORL1，是一种 I 型膜蛋白，其大量的细胞外部分，可溶性 LR11（sLR11），通过与金属蛋白酶 17（ADAM17）的分裂而被蛋白水解脱落释放。脱落机制被认为在 LR11 的功能中起着关键作用，但这方面的证据尚未得到证实。最近已经表明，四跨膜蛋白 CD9 调节肿瘤坏死因子-α和细胞间黏附分子-1在细胞表面上的 ADAM17 介导的脱落。在这里，我们研究了 CD9 在白细胞中 LR11 脱落中的作用。THP-1 单核细胞中不表达 LR11，但在佛波醇 12-肉豆蔻酸 13-醋酸酯（PMA）诱导的 THP-1 巨噬细胞（PMA/THP-1）中表达和释放。共聚焦显微镜显示 PMA/THP-1 细胞表面上 LR11 和 CD9 蛋白的共定位。在 CCFR-SB 细胞（LR11 阳性且 CD9 阴性）中异位表达 CD9 会减少从细胞中释放的 sLR11 量。相反，用中和抗 CD9 单克隆抗体孵育 LR11 转染的 THP-1 细胞会增加从细胞中释放的 sLR11 量。同样，在用金属蛋白酶抑制剂 GM6001 处理后，CD9 靶向 shRNA 转染的 PMA 刺激的 sLR11 释放增加。这些结果表明，四跨膜蛋白 CD9 调节各种白血病细胞系中 ADAM17 介导的 LR11 脱落，并且细胞表面上 LR11 和 CD9 之间的关联在 ADAM17 介导的脱落机制中起着重要作用。
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