A trans-Golgi network golgin is required for the 14 regulated secretion of TNF in activated macrophages in vivo
A trans-Golgi network golgin is required for the 14 regulated secretion of TNF in activated macrophages in vivo
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DOI：
10.1073/pnas.0800137105
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发表时间：
2008-03-04
影响因子：
11.1
通讯作者：
Gleeson, Paul A.
中科院分区：
文献类型：
--
作者：
Lieu, Zi Zhao;Lock, John G.;Gleeson, Paul A.
肿瘤坏死因子-α(TNF)的跨膜前体通过管状载体的跨高尔基网络(TGN)转运到细胞表面；然而，负责高尔基体输出的分子机制尚不清楚。我们以前报道过TGN Golgin家族的成员与TGN的亚域和小管相关。在这里，我们证明了TGN Golgin，P230/Golgin-245(P230)，在转基因的HeLa细胞和活化的巨噬细胞中，对于细胞内运输和细胞表面递送肿瘤坏死因子是必不可少的。活细胞成像显示，肿瘤坏死因子从TGN的转运是由标记为P230的小管和载体选择性地介导的。值得注意的是，巨噬细胞的内毒素激活导致从TGN涌出的P230标记的小管和载体显著增加，表明巨噬细胞上调了肿瘤坏死因子输出的运输途径。与对照细胞相比，去除内毒素刺激的巨噬细胞中的P230使细胞表面传递的肿瘤坏死因子减少了10倍。为了确定P230的缺失是否在体内阻断了肿瘤坏死因子的分泌，我们产生了表达沉默P230的microRNA载体的逆转录小鼠。用含有microRNA的重组逆转录病毒转导骨髓干细胞，并将其移植到受照射的受者体内。来自P230miRNA逆转录小鼠的脂多糖激活的腹膜巨噬细胞耗尽了P230，并显著降低了细胞表面肿瘤坏死因子的水平。总体而言，这些研究已确定P230是肿瘤坏死因子分泌的关键调节因子，并表明巨噬细胞的嘴唇激活导致出口高尔基体携带者增加。此外，我们还展示了一种以前没有描述过的方法，通过对贩运机制的特定沉默来控制细胞因子的分泌。
The transmembrane precursor of tumor necrosis factor-a (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 LIPS 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.