摘要/Abstract

摘要： 目的·探讨过氧化物酶体增殖物激活受体γ1（peroxisome proliferator activated receptor γ1，PPARγ1）的SUMO化修饰在白介素4（interleukin-4，IL-4）诱导巨噬细胞M2极化过程中的作用和机制。方法·在HEK293T细胞中共转染FLAG-PPARγ1-WT/突变体质粒、HA-SUMO1质粒，使用标签FLAG及HA的抗体分别进行免疫共沉淀后进行蛋白质印迹分析，确认PPARγ1的SUMO化修饰和修饰位点。过表达FLAG-PPARγ1-WT、HA-SUMO1及野生型RGS-SENP1以及去SUMO化修饰酶活缺失的突变体RGS-SENP1mut，证明SENP1是PPARγ1的去SUMO化修饰酶。用IL-4刺激小鼠巨噬细胞系RAW264.7和体外分离的原代小鼠腹腔巨噬细胞，使其向M2型活化，使用PPARγ及SUMO1的抗体进行免疫共沉淀，明确内源性PPARγ1的SUMO化修饰在M2极化过程中的变化。采用实时荧光定量聚合酶链反应检测IL-4刺激后的PPARγ1野生型及突变型稳转细胞系RAW264.7中M2相关基因的mRNA水平。用染色质免疫共沉淀实验比较PPARγ1 WT及突变体结合于精氨酸酶Ⅰ（arginaseⅠ，Arg1）启动子的能力。结果·PPARγ1蛋白第77位赖氨酸（K77R）能被SUMO化修饰，并能被SENP1去SUMO化。在IL-4诱导巨噬细胞M2型极化时，PPARγ1的SUMO化水平降低。稳定表达SUMO化位点突变体PPARγ1-K77R的RAW264.7细胞，Arg1 mRNA 水平升高；PPARγ1-K77R与Arg1的启动子结合能力增强，促进Arg1表达。结论·PPARγ1通过去SUMO化促进下游Arg1基因的表达从而参与调控巨噬细胞M2型极化。
关键词: 过氧化物酶体增殖物激活受体&, gamma, 1, SUMO化修饰, SENP1, 巨噬细胞, M2极化
Abstract:
Objective · To investigate the role and the regulation mechanism of SUMOylation of peroxisome proliferator activated receptor γ1 (PPARγ1) in macrophage M2 polarization inducedinterleukin-4 (IL-4). Methods · To investigate the SUMOylation of PPARγ1 and identify its SUMOylated site, immunoprecipitation (IP) with anti-FLAG/HA antibody and Western blotting were used after plasmids FLAG-PPARγ1-WT/mutant and HA-SUMO1 being co-transfected into HEK293T cells. To determine SENP1 can de-SUMOylate PPARγ1, IP was used when HEK293T cells were co-transfectedFLAG-PPARγ1-WT, HA-SUMO1 and RGS-SENP1-WT, or SENP1 mutant plasmids. The change of the endogenous SUMOylation of PPARγ1 during M2 polarization was checkedIP and Western blottingusing PPARγ or SUMO1 antibodies in cell lysates of RAW264.7 cells and primary peritoneal macrophages inducedIL-4. The of some M2 related marker genes were detectedreal-time quantitative polymerase chain reaction in PPARγ1-WT/mutants stably-overexpressed RAW264.7 cells. Chromatin immunoprecipitation (ChIP) experiment was used to confirm the different ability of binding to the promoter of arginaseⅠ (Arg1) between PPARγ1-WT and PPARγ1-K77R. Results · It has been identified that the major SUMOylated site of PPARγ1 was Lys77, which could be de-SUMOylatedSENP1. The endogenous SUMOylation of PPARγ1 decreased when macrophage polarized to M2 macrophage inducedIL-4. The of Arg1 increased in PPARγ1-K77R stably-overexpressed RAW264.7 cells. PPARγ1-K77R easily bound to the promoter of Arg1 gene, showing more transcription activity. Conclusion · De-SUMOylation of PPARγ1 at Lys77 can enhance its transcription activitypromoting the of Arg1 gene, which is involved in the regulation of macrophage M2 polarization.
Key words: peroxisome proliferator activated receptor γ1 (PPARγ1), SUMOylation, SENP1, macrophage, M2 polarization


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