铁离子参与自身免疫性疾病中促炎性细胞因子的转录后调控机制研究

Iron deposition is frequently observed in human autoinflammatory diseases, such as in the central nervous system (CNS) of patients with multiple sclerosis or in the blood of patients with rhemeotoid arthritis. Yet, the functional outcome of excessive iron in the auto-inflammatory response is largely unknown. Granulocyte macrophage colony-stimulating factor (GM-CSF) is a proinflammatory cytokine promoting myeloid cell maturation and activation, which are essential for the pathogenesis of many autoimmune diseases. GM-CSF expression is regulated by its 3’ untranslated region (3’UTR) via interaction with specific RNA-binding proteins. In a targeted short hairpin (sh) RNA screen, we have identified that Poly(rC) binding protein 1 (PCBP1), an RNA binding protein with iron chaperon function, enhanced GM-CSF 3’UTR activity and mRNA stability. PCBP1 deficiency in autoreactive T cells resulted in compromised production of GM-CSF and other cytokines, abolishing their capacity in inducing experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. We further showed that ferrous iron was required for the expression and activity of PCBP1, as iron depletion degraded PCBP1 protein and suppressed GM-CSF production. These results suggest a model that excessive iron may directly precipitate autoimmune diseases through post-transcriptional regulation of pro-inflammatory cytokine expression. .In the proposed study, we will identify transcriptome-wide targets of PCBP1, and systemically examine how iron metabolism regulates pro-inflammatory cytokine expression through PCBP1-mediated RNA stabilization; by analyzing PCBP1 deficient and Transferrin receptor (TfR) deficient mice, we will examine iron-PCBP1 mediated post-transcription mechanisms in the pathogenesis of rheumatoid arthritis; Finally, we will analyze interplay among PCBP1 expression, iron deposition and proinflammatory cytokine expression in patients with rheumatoid arthritis. .Our proposed studies will not only elucidate new regulatory mechanisms governing proinflammatory cytokine expression, but also provide considerable new insights into the molecular pathogenesis of rheumatoid arthritis. Knowledge gained through this project is anticipated to expedite the development of effective therapies to treat rheumatoid arthritis by targeting iron homeostasis. Although we propose a study specific to rheumatoid arthritis, the concepts and innovative/tools/reagents developed in this project can readily be broadened to encompass other similar autoimmune disorders.

自身免疫性疾病是免疫系统错误识别自身组织，产生免疫应答和过量促炎性细胞因子，造成组织损伤的疾病。自身免疫疾病经常伴随铁离子的异常富集。前期高通量shRNA筛选发现，具有铁离子伴侣功能的RNA结合蛋白PCBP1，可以通过调节GM-CSF等细胞因子mRNA稳定性，促进其在T细胞中的表达，诱导小鼠实验性自身免疫脑脊髓炎（EAE）发病；铁离子调节PCBP1的蛋白稳定性，在转录后水平促进细胞因子的分泌。我们提出科学假说：在自身免疫性疾病中，铁离子稳定PCBP1蛋白，促进细胞因子的产生，加剧免疫反应，直接参与自身免疫性疾病。本项目将通过PAR-CLIP等高通量方法，鉴定PCBP1在转录组水平上的靶点mRNA; 利用基因敲除小鼠，分析铁-PCBP1介导的转录后调控机制，在类风湿性关节炎等自身免疫疾病中的作用。本项目将建立铁离子通过转录后调控参与免疫应答的机制,从而筛选出新的针对自身免疫性疾病的治疗靶点。

在真核细胞中，RNA在转录后受多种不同机制的调节，包括不同剪切方式，3’聚腺苷酸化，microRNA等。这些机制亦受到相应的RNA结合蛋白（RBP）调控：例如，RNA的剪切蛋白，可以通过不同的方式剪接RNA，从而使同一基因产生多个转录子及相应蛋白质产物。.我们在高通量shRNA筛选发现，具有铁离子伴侣功能的RNA结合蛋白PCBP1，可以通过调节GM-CSF等细胞因子mRNA稳定性，促进其在T细胞中的表达，诱导小鼠实验性自身免疫脑脊髓炎（EAE）发病；通过PAR-CLIP等高通量方法，鉴定出PCBP1在转录组水平上的靶点mRNA，证明PCBP1通过结合到RNA 3’UTR中的Poly(rC)元件，调节细胞因子mRNA的稳定性。.同时，我们发现作为铁离子伴侣蛋白，胞内铁离子浓度可以调节PCBP1的蛋白质稳定性，在转录后水平促进细胞因子的分泌。无论利用铁离子敖合剂或基因缺陷小鼠改变T细胞中铁离子含量，都会影响促炎性细胞因子（如GM-CSF，IL2等）的表达，进而影响多样性硬化的小鼠模型EAE的发生。更为重要的是，铁离子对促炎性细胞因子的调控主要在转录后水平，通过影响促炎性细胞因子的mRNA的稳定性来实现。铁离子保护PCBP1蛋白不被Caspase剪切降解，增强靶基因mRNA的稳定性、促进促炎性细胞因子的产生, 提示PCBP1可以作为胞内铁离子的传感器，调控免疫细胞转录组的动态变化和致病性。.该研究鉴定出调控GM-CSF表达的新的顺式作用元件，及其相关的反式作用因子PCBP1，揭示了GM-CSF新的转录后调控机制；同时丰富了代谢环境通过RNA结合蛋白转录后调控基因表达的模型；阐释了铁代谢异常促进自身免疫疾病的机制，为多发性硬化等自身免疫疾病的治疗提供的新的靶点和思路。