La Xiaoqin, Zhang Lichao, Li Hanqing, Li Zhuoyu, Song Guisheng, Yang Peng, Yang Yufei
Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of National Ministry of Education, Shanxi University, Taiyuan 030006, China.
Institute of Biomedical Sciences, Shanxi University, Taiyuan 030006, China.
Oncotarget. 2018 Jan 9;9(21):15464-15479. doi: 10.18632/oncotarget.24090. eCollection 2018 Mar 20.
Glucose-regulated protein 78 (GRP78), an ER chaperone, is overexpressed in cancer cells. Solid tumor cells can secrete GRP78 that can promote tumor angiogenesis, differentiation of bone marrow-derived mesenchymal stem cells, tumor cell proliferation and polarization of tumor-associated macrophages. However, the mechanism by which GRP78 functions as a tumor promoter either by staying on the membrane to stimulate intracellular signals or directly entering into cytosolic remains unknown. Here, we reported that an endotoxin-free His-GRP78 protein was purified that simulates original secreted GRP78. Through analyzing GRP78 concentration in serum samples from 32 colon cancer patients, 40 nM His-GRP78 was selected as an optimized dose to treat cells. Biochemical analysis revealed that secreted GRP78 was able to enter into RAW264.7 and THP-1 cells directly rather than stay on the plasma membrane to transfer signals. Further studies showed that GRP78 internalization was endocytosis-dependent, and both phagocytosis and clathrin, caveolin-1 and micropinocytosis-mediated endocytosis pathways contributed to internalization of secreted GRP78 into cells. Mechanistically, Ajuba is able to interact with GRP78. Ablation of Ajuba suppressed the internalization of secreted GRP78 into cells, indicating that Ajuba was responsible for internalization of secreted GRP78 into RAW264.7. Furthermore, we observed that internalized GRP78 could entered into the mitochondrion and endoplasmic reticulum, which provided a suitable place and enough time for GRP78 to function in molecular and cellular processes. Together, these results reveal a novel mechanism by which secreted GRP78 internalizes into macrophages in the tumor microenvironment, which provides a potential target for drug development.
葡萄糖调节蛋白78（GRP78）是一种内质网伴侣蛋白，在癌细胞中过表达。实体瘤细胞可分泌GRP78，其能够促进肿瘤血管生成、骨髓间充质干细胞分化、肿瘤细胞增殖以及肿瘤相关巨噬细胞极化。然而，GRP78作为肿瘤促进因子，是通过留在细胞膜上刺激细胞内信号还是直接进入细胞质发挥作用的机制尚不清楚。在此，我们报道了一种无内毒素的His-GRP78蛋白被纯化出来，其模拟原始分泌的GRP78。通过分析32例结肠癌患者血清样本中的GRP78浓度，选择40 nM His-GRP78作为处理细胞的优化剂量。生化分析表明，分泌的GRP78能够直接进入RAW264.7和THP-1细胞，而不是留在质膜上传递信号。进一步研究表明，GRP78的内化依赖于内吞作用，吞噬作用以及网格蛋白、小窝蛋白-1和微胞饮作用介导的内吞途径均有助于分泌的GRP78内化进入细胞。机制上，Ajuba能够与GRP78相互作用。敲除Ajuba可抑制分泌的GRP78内化进入细胞，表明Ajuba负责分泌的GRP78内化进入RAW264.7细胞。此外，我们观察到内化的GRP78能够进入线粒体和内质网，这为GRP78在分子和细胞过程中发挥作用提供了合适的场所和充足的时间。总之，这些结果揭示了一种新的机制，即分泌的GRP78在肿瘤微环境中内化进入巨噬细胞，这为药物开发提供了潜在靶点。
