巨噬细胞通过炎症因子级联放大效应增加子宫内膜雌激素敏感性的机制

Persistant estrogen stimulation causes unregulated endometrial proliferation. Studies showed that macrophages could up-regulate estrogen receptor α (ERα) and thus enhanced estrogen sensitivity in endometrium. This might be one of the important mechanisms for unregulated endometrial proliferation. But the mechanism through which macrophages up-regulate ERα is still unclear. We found that endometrial microenvironment could induce inflammatory cytokines production in macrophages and thus up-regulate ERα expression in endometrial cells. Ubiquitin-modifying ERα could affect stability of ERα protein as well as transcriptional activity of its downstream target genes. Ubiquitin-modifying protein A20 was strongly up-regulated in endometrial hyperplasia mouse model. We thus suppose that the inflammatory cytokines produced by infiltrating macrophages in endometrium could up-regulate their own production through positive feedback regulation. These cytokines collabrately increased endometrial estrogen sensitivity through promoting ERα transcription; ubiquitinating ERα protein through A20, which resulted in stabilizing ERα protein and promoting transcription of its downstream target genes. These worked synergistically to amplify the effect of enhanced estrogen sensitivity in endometrium. We will testify our hypothesis by in vitro study and animal experiments. We will analyze the positive feedback of cytokines produced by macrophages and the synergistic effect of cytokines on promoting ERα expression; investigate the effect and mechanisms of cytokines on ERα protein stability and its downstream target genes transcriptional activity through ubiquitinating ERα protein by A20; testify the synergistic effect of cytokines on enhancing estrogen sensitivity in endometrial cells. The study will help the prevention and treatment of endometrial hyperplasia.

持续雌激素刺激导致子宫内膜异常增殖。研究发现巨噬细胞上调子宫内膜雌激素受体α（ERα）增加局部雌激素敏感性是内膜异常增殖的重要机制，但巨噬细胞如何调控ERα并不清楚。我们发现内膜微环境可诱导巨噬细胞炎症因子释放进而上调内膜细胞ERα表达。泛素修饰ERα可影响其蛋白稳定性及下游靶基因转录活性。内膜增生小鼠模型显示泛素修饰蛋白A20显著上调。由此推测巨噬细胞很可能通过正反馈调控释放大量炎症因子，后者协同作用促进ERα转录，并通过A20泛素修饰ERα蛋白，稳定ERα表达同时促进其下游靶基因转录，导致内膜对雌激素敏感性增加。为验证该假说，本项目通过体外和动物实验分析巨噬细胞炎症因子的正反馈调控及上调内膜ERα表达的协同效应；探索炎症因子通过A20泛素修饰ERα对其蛋白稳定性及下游靶基因转录活性的调控作用及泛素修饰机制，验证炎症因子调控内膜细胞雌激素敏感性的协同作用。为防治内膜异常增殖提供依据。

无孕激素保护的雌激素长期暴露是子宫内膜异常增殖和子宫内膜癌发生的主要机制。然而，与健康对照相比，多数子宫内膜增生性病变及子宫内膜癌患者血清雌激素水平未见升高，提示子宫内膜局部雌激素敏感性可能是驱动子宫内膜异常增殖和子宫内膜癌发生的关键。我们前期研究发现CD163+巨噬细胞可以促进雌激素依赖性EC的发展，但其机制尚不清楚。流式细胞学分析显示CD163+巨噬细胞是子宫内膜非典型增生和子宫内膜癌组织中占主导地位的巨噬细胞，其浸润与ERα表达呈正相关。 CD163+巨噬细胞主要增加ERα蛋白水平，但对ESR1（ERα编码基因）转录本的上调作用很小。小鼠子宫内膜表达谱差异基因分析发现，高脂饮食和雌激素暴露的小鼠子宫内膜泛素编辑酶A20显著升高。进一步免疫组化分析发现子宫内膜增生性病变及子宫内膜样癌（G1）组织中CD163+巨噬细胞浸润增多，且A20与ERα蛋白表达呈正相关。体外研究发现CD163+巨噬细胞可上调子宫内膜癌细胞A20与ERα蛋白表达。同样，CD163+巨噬细胞通过细胞因子如IL1α，IL17A和TNFα上调A20和ERα蛋白水平，但对ERα转录水平影响较小。机制上，A20仅正向调控ERα蛋白翻译水平，而对ESR1转录水平和ESR1启动子区域无激活作用；应用蛋白合成抑制剂CHX后，A20可显著延长ERα蛋白半衰期，提示A20可能通过阻止ERα蛋白降解以稳定ERα蛋白。蛋白免疫共沉淀、细胞免疫荧光数据显示A20可与ERα蛋白在细胞核中形成复合物以稳定ERα蛋白。构建A20截短体和突变体，Western Blotting、泛素实验等显示A20可通过其OTU结构域的去泛素化酶活性降低ERα去泛素化修饰从而稳定ERα蛋白。体内外实验显示过表达A20可上调功能性ERα蛋白从而增加子宫内膜癌细胞对雌激素的敏感性。针对慢性炎症微环境在子宫内膜癌发病机制中的作用，本研究深入探讨了CD163+巨噬细胞相关的慢性炎症微环境下，ERα蛋白的异常泛素化修饰与雌激素依赖相关肿瘤发生机制。该研究有利于深入理解CD163+巨噬细胞相关慢性炎症调控子宫内膜雌激素敏感性的作用机制，并为临床子宫内膜癌的早期诊断和治疗新方法提供方向。