Supplementary Material for: Degradation of Ubiquitin-Editing Enzyme A20 following Autophagy Activation Promotes RNF168 Nuclear Translocation and NF-κB Activation in Lupus Nephritis

The correlation between ubiquitin-editing enzyme A20 and E3 ubiquitin ligase ring finger protein (RNF) 168 has been reported to be critical for repair of DNA damage. This study aimed to evaluate the potential role of this regulatory interaction in the pathogenesis of lupus nephritis (LN). The expression of RNF168 and A20 was measured in the podocytes derived from MRL/lpr murine lupus as well as patients with LN. Cell-based studies using renal podocytes bearing silenced RNF168, over-expressed A20, autophagy-related gene (Atg) 5 (a ubiquitin-like modifier), or silenced Atg5 were used to assess the effect of RNF168, A20, and Atg5 on DNA damage repair and nuclear factor kappa-B (NF-κB) activation in LN. It was found that podocyte autophagy was over-activated in LN and the abnormal podocyte autophagy led to down-regulation of A20, up-regulation of RNF168, and activation of the NF-κB. RNF168 silencing or A20 restoration inhibited activation of NF-κB pathway and promoted repair of DNA damage, where the level of autophagy was not changed. Activated A20 in podocytes weakened the promoting action of cell autophagy on RNF168. The current results suggest that RNF168 dysfunction may be involved in the pathogenesis of LN via down-regulation of A20 expression. Autophagy and RNF168 may be therapeutic targets for the prevention and treatment of LN.

已有研究表明，泛素编辑酶A20（ubiquitin-editing enzyme A20）与E3泛素连接酶环指蛋白168（Ring Finger Protein 168, RNF168）之间的调控互作对DNA损伤修复至关重要。本研究旨在探讨该调控互作在狼疮肾炎（Lupus Nephritis, LN）发病机制中的潜在作用。研究人员检测了MRL/lpr小鼠狼疮模型来源的足细胞以及狼疮肾炎患者足细胞中RNF168与A20的表达水平。通过对肾足细胞进行RNF168沉默、A20过表达、自噬相关基因5（Autophagy-related Gene 5, Atg5，一种泛素样修饰蛋白）沉默或过表达等基于细胞的实验，评估RNF168、A20及Atg5对狼疮肾炎中DNA损伤修复与核因子κB（Nuclear Factor Kappa-B, NF-κB）活化的影响。研究发现，狼疮肾炎患者足细胞的自噬存在过度激活现象，异常的足细胞自噬会导致A20表达下调、RNF168表达上调并激活核因子κB通路。沉默RNF168或恢复A20表达，可抑制核因子κB通路活化并促进DNA损伤修复，且该过程不改变自噬水平。足细胞中活化的A20可削弱细胞自噬对RNF168的促进作用。本研究结果提示，RNF168功能异常可能通过下调A20的表达参与狼疮肾炎的发病过程，自噬与RNF168或可作为狼疮肾炎防治的潜在治疗靶点。