Centre for Experimental Medicine and Rheumatology, Queen Mary University of London, London, UK.
Centre for Experimental Medicine and Rheumatology, Queen Mary University of London, London, UK.
Lancet. 2015 Feb 26;385 Suppl 1:S9. doi: 10.1016/S0140-6736(15)60324-5.
A single risk haplotype across UBE2L3 is strongly associated with systemic lupus erythematosus (SLE) and many other autoimmune diseases. UBE2L3 is an E2 ubiquitin-conjugating enzyme with specificity for RING-in-between-RING E3 ligases, including HOIL-1 and HOIP, components of the linear ubiquitin chain assembly complex (LUBAC), which has a pivotal role in inflammation, through crucial regulation of NF-κB. We aimed to determine whether UBE2L3 regulates LUBAC-mediated activation of NF-κB, and determine the effect of UBE2L3 genotype on NF-κB activation and B-cell differentiation.
UBE2L3 genotype data from SLE genome-wide association studies was imputed by use of 1000 Genomes data. UBE2L3 function was studied in a HEK293-NF-κB reporter cell line with standard molecular biology techniques. p65 NF-κB translocation in ex-vivo B cells and monocytes from genotyped healthy individuals was quantified by imaging flow cytometry. B-cell subsets from healthy individuals and patients with SLE, stratified by UBE2L3 genotype, were determined by multicolour flow cytometry.
rs140490, located at -270 base pairs of the UBE2L3 promoter, was identified as the most strongly associated single nucleotide polymorphism (p=8·6 × 10(-14), odds ratio 1·30, 95% CI 1·21-1·39). The rs140490 risk allele increased UBE2L3 expression in B cells and monocytes. Marked upregulation of NF-κB was observed with combined overexpression of UBE2L3 and LUBAC, but abolished by dominant-negative mutant UBE2L3 (C86S), or UBE2L3 silencing. The rs140490 genotype correlated with basal NF-κB activation in ex-vivo human B cells and monocytes, as well as NF-κB sensitivity to CD40 or tumour necrosis factor (TNF) stimulation. UBE2L3 expression was 3-4 times higher in circulating plasmablasts and plasma cells than in other B-cell subsets, with higher levels in patients with SLE than in controls. The rs140490 genotype correlated with increasing plasmablast and plasma cell differentiation in patients with SLE.
This study shows that NF-κB activation mediated by LUBAC is exquisitely sensitive to the expression level of UBE2L3. The UBE2L3 risk haplotype is correlated with TNF and CD40 induced NF-κB activation in primary human cells, and with plasmablast and plasma cell expansion in SLE, consistent with the dependence of these cells on NF-κB as a survival factor. Since UBE2L3 is highly expressed in plasma cells, UBE2L3 could be a novel therapeutic target in SLE.
Arthritis Research UK, Wellcome Trust, George Koukis Foundation, European Community's Seventh Framework Programme.
UBE2L3 上的单个风险单倍型与系统性红斑狼疮 (SLE) 和许多其他自身免疫性疾病密切相关。UBE2L3 是一种 E2 泛素缀合酶，特异性识别 RING-in-between-RING E3 连接酶，包括 HOIL-1 和 HOIP，它们是线性泛素链组装复合物 (LUBAC) 的组成部分，在炎症中发挥关键作用，通过对 NF-κB 的关键调节。我们旨在确定 UBE2L3 是否调节 LUBAC 介导的 NF-κB 激活，并确定 UBE2L3 基因型对 NF-κB 激活和 B 细胞分化的影响。
使用 1000 基因组数据对 SLE 全基因组关联研究中的 UBE2L3 基因型数据进行了推断。使用标准分子生物学技术在 HEK293-NF-κB 报告细胞系中研究了 UBE2L3 的功能。通过成像流式细胞术定量测定了来自基因分型健康个体的 ex-vivo B 细胞和单核细胞中的 p65 NF-κB 易位。通过多色流式细胞术确定了来自健康个体和 SLE 患者的 B 细胞亚群，这些个体根据 UBE2L3 基因型进行了分层。
位于 UBE2L3 启动子-270 个碱基对的 rs140490 被确定为最强烈相关的单核苷酸多态性 (p=8.6 × 10(-14)，优势比 1.30，95%CI 1.21-1.39)。rs140490 风险等位基因增加了 B 细胞和单核细胞中的 UBE2L3 表达。观察到 UBE2L3 和 LUBAC 的联合过表达会明显上调 NF-κB，但用显性负突变体 UBE2L3 (C86S) 或 UBE2L3 沉默会消除这种上调。rs140490 基因型与 ex-vivo 人类 B 细胞和单核细胞中 NF-κB 的基础激活以及 NF-κB 对 CD40 或肿瘤坏死因子 (TNF) 刺激的敏感性相关。UBE2L3 在循环浆母细胞和浆细胞中的表达水平比其他 B 细胞亚群高 3-4 倍，SLE 患者中的表达水平高于对照组。rs140490 基因型与 SLE 患者中浆母细胞和浆细胞分化的增加相关。
这项研究表明，LUBAC 介导的 NF-κB 激活对 UBE2L3 的表达水平非常敏感。UBE2L3 风险单倍型与 TNF 和 CD40 诱导的原发性人细胞中的 NF-κB 激活以及 SLE 中的浆母细胞和浆细胞扩增相关，这与这些细胞依赖 NF-κB 作为生存因子一致。由于 UBE2L3 在浆细胞中高度表达，UBE2L3 可能成为 SLE 的一种新的治疗靶点。
关节炎研究英国，惠康信托基金会，乔治·科基斯基金会，欧盟第七框架计划。
