Dorn Sebastian, Schoergenhofer Christian, Krainer Michael, Müller Markus, Jilma Bernd
Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria.
Clinical Division of Oncology, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria.
Biores Open Access. 2018 May 1;7(1):81-89. doi: 10.1089/biores.2018.0006. eCollection 2018.
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known to activate the canonical NF-κB pathway similar to TNF. The exact mechanism of the entire signaling cascade is still under investigation. The involvement of linear ubiquitylation as upregulating component has already been shown recently in some cell lines, but not in human embryonic kidney 293 (HEK293) cells. The downregulating function of the ABIN-1 (A20 binding and inhibitor of NF-κB) as linear ubiquitylation antagonist has been shown in combination with some NF-κB-inducing pathways, but not with TRAIL. We performed luciferase and western blot assays using HEK293 cells stimulated with either TRAIL (or TNF as a control) to analyze the involvement of linear ubiquitin chain assembly complex (LUBAC) components and the impact of ABIN-1 and ABIN-1-MAD (truncated form without A20 binding site) on NF-κB signaling. For overexpression experiments, we added plasmids of ABIN-1 and ABIN-1-MAD or LUBAC components HOIP, HOIL-1, or SHARPIN (single and combinations). For downregulation experiments five pairs of either SHARPIN, HOIL-1, or HOIP targeting miRNAs or one miRNA for ABIN-1 were designed and added. ABIN-1 and its truncated form ABIN-1-MAD reduced the NF-κB induction significantly indicating its involvement as antagonist (independent of deubiquitinase A20) of linear ubiquitylation in TRAIL-induced NF-κB signaling. In opposition, knockdown of ABIN-1 using a specific ABIN-1 miRNA led a clear increase of NF-κB signaling. Addition of single LUBAC components or combinations (except for SHARPIN with HOIL-1) resulted in clearly stronger NF-κB inductions. MiRNAs targeting LUBAC components significantly reduced NF-κB activation. Thus, in HEK293 cells linear ubiquitylation by LUBAC critically upregulates and ABIN-1 downregulates TRAIL-induced NF-κB signaling and may be interesting targets for future pathological therapies.
已知肿瘤坏死因子（TNF）相关凋亡诱导配体（TRAIL）与TNF类似，可激活经典的NF-κB信号通路。整个信号级联的确切机制仍在研究中。线性泛素化作为上调成分的作用最近已在一些细胞系中得到证实，但在人胚肾293（HEK293）细胞中尚未得到证实。ABIN-1（A20结合蛋白和NF-κB抑制剂）作为线性泛素化拮抗剂的下调功能已在某些NF-κB诱导途径中得到证实，但在TRAIL诱导途径中尚未得到证实。我们使用经TRAIL（或作为对照的TNF）刺激的HEK293细胞进行荧光素酶和蛋白质印迹分析，以分析线性泛素链组装复合物（LUBAC）成分的作用以及ABIN-1和ABIN-1-MAD（无A20结合位点的截短形式）对NF-κB信号传导的影响。在过表达实验中，我们添加了ABIN-1和ABIN-1-MAD或LUBAC成分HOIP、HOIL-1或SHARPIN（单独及组合）的质粒。在下调实验中，设计并添加了五对靶向SHARPIN、HOIL-1或HOIP的miRNA或一对靶向ABIN-1的miRNA。ABIN-1及其截短形式ABIN-1-MAD显著降低了NF-κB的诱导，表明其作为TRAIL诱导的NF-κB信号传导中线性泛素化的拮抗剂（独立于去泛素酶A20）发挥作用。相反，使用特异性ABIN-1 miRNA敲低ABIN-1导致NF-κB信号明显增强。添加单个LUBAC成分或组合（SHARPIN与HOIL-1组合除外）导致NF-κB诱导明显增强。靶向LUBAC成分的miRNA显著降低了NF-κB激活。因此，在HEK293细胞中，LUBAC介导的线性泛素化关键上调TRAIL诱导的NF-κB信号，而ABIN-1下调该信号，它们可能是未来病理治疗的有趣靶点。
