Lieu Zi Zhao, Lock John G, Hammond Luke A, La Gruta Nicole L, Stow Jennifer L, Gleeson Paul A
Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute and Department of Microbiology and Immunology, University of Melbourne, Victoria 3010, Australia.
Proc Natl Acad Sci U S A. 2008 Mar 4;105(9):3351-6. doi: 10.1073/pnas.0800137105. Epub 2008 Feb 28.
The transmembrane precursor of tumor necrosis factor-alpha (TNF) exits the trans-Golgi network (TGN) in tubular carriers for subsequent trafficking and delivery to the cell surface; however, the molecular machinery responsible for Golgi export is unknown. We previously reported that members of the TGN golgin family are associated with subdomains and tubules of the TGN. Here, we show that the TGN golgin, p230/golgin-245 (p230), is essential for intracellular trafficking and cell surface delivery of TNF in transfected HeLa cells and activated macrophages. Live-cell imaging revealed that TNF transport from the TGN is mediated selectively by tubules and carriers marked by p230. Significantly, LPS activation of macrophages resulted in a dramatic increase of p230-labeled tubules and carriers emerging from the TGN, indicating that macrophages up-regulate the transport pathway for TNF export. Depletion of p230 in LPS-stimulated macrophages reduced cell surface delivery of TNF by >10-fold compared with control cells. To determine whether p230 depletion blocked TNF secretion in vivo, we generated retrogenic mice expressing a microRNA-vector to silence p230. Bone-marrow stem cells were transduced with recombinant retrovirus containing microRNA constructs and transplanted into irradiated recipients. LPS-activated peritoneal macrophages from p230 miRNA retrogenic mice were depleted of p230 and had dramatically reduced levels of cell surface TNF. Overall, these studies have identified p230 as a key regulator of TNF secretion and have shown that LPS activation of macrophages results in increased Golgi carriers for export. Also, we have demonstrated a previously undescribed approach to control cytokine secretion by the specific silencing of trafficking machinery.
肿瘤坏死因子-α（TNF）的跨膜前体通过管状载体离开反式高尔基体网络（TGN），以便随后运输并递送至细胞表面；然而，负责高尔基体输出的分子机制尚不清楚。我们之前报道过，TGN golgin家族成员与TGN的亚结构域和小管相关。在此，我们表明TGN golgin，p230/golgin-245（p230）对于转染的HeLa细胞和活化的巨噬细胞中TNF的细胞内运输和细胞表面递送至关重要。活细胞成像显示，TNF从TGN的运输由以p230标记的小管和载体选择性介导。值得注意的是，巨噬细胞的LPS激活导致从TGN出现的p230标记的小管和载体显著增加，表明巨噬细胞上调了TNF输出的运输途径。与对照细胞相比，LPS刺激的巨噬细胞中p230的缺失使TNF的细胞表面递送减少了10倍以上。为了确定p230的缺失是否在体内阻断了TNF的分泌，我们生成了表达微小RNA载体以沉默p230的逆转基因小鼠。用含有微小RNA构建体的重组逆转录病毒转导骨髓干细胞，并将其移植到受辐照的受体中。来自p230 miRNA逆转基因小鼠的LPS激活的腹膜巨噬细胞中p230缺失，且细胞表面TNF水平显著降低。总体而言，这些研究确定p230是TNF分泌的关键调节因子，并表明巨噬细胞的LPS激活导致用于输出的高尔基体载体增加。此外，我们展示了一种先前未描述的通过特异性沉默运输机制来控制细胞因子分泌的方法。
