E3泛素连接酶NEDD4L对IL-1信号转导及溃疡性结肠炎的调节作用

Interleukin-1 (IL-1) signaling pathway is associated with the pathogenesis of inflammatory bowel disease (IBD) by promoting inflammatory mediators. E3 ubiquitin ligase and deubiquitinase play a crucial role in the regulation of IL-1 signaling. Neural precursor cell expressed, developmentally down-regulated gene 4-like (NEDD4L) is one of the E3 ubiquitin ligases. It was found recently that NEDD4L expression was reduced in the tissue of colorectal cancer. Furthermore, it was found that the level of NEDD4L expression is associated with the prognosis of patients with digestive cancer and non-small cell lung cancer. We found, by GEO data analysis, that the expression of NEDD4L was significantly decreased in colon samples from patients with ulcerative colitis. Our in vitro experiments showed that NEDD4L negatively control IL-1 induced inflammation through inhibiting MEKK2 expression. In this program, we are going to verify the aforementioned conclusion and observe IL-1 induced inflammation in cells derived from NEDD4L deficient mice, to investigate the regulatory effects of NEDD4L on IL-1 mediated signaling pathway and the related molecular mechanisms, to clarify the target molecules through which NEDD4L regulates IL-1 mediated signaling, to study the regulatory role of NEDD4L in the pathogenesis of IBD. Our results will provide novel insights into the regulation of IL-1 signaling pathway and the pathogenesis of IBD and may also provide a potential therapeutic target for IBD.

IL-1信号通路促进炎症介质产生，与炎症性肠病（IBD）的发生发展密切相关。E3泛素连接酶和去泛素化酶在IL-1信号通路中具有重要的调控作用。NEDD4L是进化上高度保守的一种E3泛素连接酶，近年发现结直肠癌组织中NEDD4L表达下调，并且与消化道肿瘤和非小细胞肺癌预后相关。我们通过GEO数据分析发现，在溃疡性结肠炎患者结肠组织中，NEDD4L表达显著下调。体外实验提示NEDD4L通过抑制MEKK2表达负向调控IL-1诱导的炎症反应。本项目拟利用NEDD4L基因缺陷小鼠肠炎模型，验证前期实验结论，观察该缺陷小鼠细胞IL-1诱导的炎症反应水平；探究NEDD4L在IL-1下游信号网络中的调控作用和分子机制，阐明NEDD4L作用的靶分子及其相互作用方式；明确NEDD4L在IBD发生中的作用。为进一步阐明IBD的发生机制提供新的实验依据，为IBD的治疗提供新的作用靶点。

炎症性肠病（IBD）是严重影响人类生活质量的免疫相关病，前期研究发现，IBD患者结肠黏膜组织中NEDD4L水平下降。本项目以Nedd4l基因缺陷小鼠及其同窝对照小鼠为主要研究材料，通过葡聚糖硫酸钠(DSS)诱导构建肠炎小鼠模型。观察小鼠体重、结肠长度、结肠出血程度和上皮损伤程度的差异，明确NEDD4L在炎症性肠病发生中的作用。以实时定量 PCR、ELISA方法，检测Nedd4l缺陷小鼠及其同窝对照小鼠及该肠炎模型的结肠组织中IL-1、IL-6、CSF3、CCL20的表达水平，探究NEDD4L调控炎症性肠病的机制。我们的结果显示：与Nedd4l+/+组相比，Nedd4l+/-组小鼠体重显著减轻，结肠出血显著加重，结肠长度显著缩短，结肠黏膜损伤显著加重，炎症细胞浸润显著增多，Nedd4l+/-组结肠组织中IL-1b、IL-6、CSF3、CCL20 mRNA和蛋白水平显著增高。这些结果表明，Nedd4l+/-能显著加重DSS诱导的小鼠IBD疾病活动度和结肠黏膜局部的病理损伤，提示NEDD4L对DSS诱导的小鼠IBD的发生具有负向调控作用。.此外，我们在前期发现（即分泌含TIR结构域的蛋白——TcpC的UPEC CFT073野生株，抑制巨噬细胞内MyD88水平和巨噬细胞固有免疫功能）的基础上，进一步观察基因重组TcpC对巨噬细胞MyD88蛋白水平和mRNA水平的影响，结果显示：TcpC能剂量和时间依赖性地抑制巨噬细胞MyD88蛋白水平，而对其mRNA水平无显著影响，并促进MyD88在蛋白酶体的积聚，提示TcpC促进MyD88降解。生物信息学分析显示：TcpC含有HECT家族E3泛素连接酶的结构特征，而人和小鼠MyD88中含有HECT家族E3泛素连接酶底物所含有的P-Y基序，提示TcpC是一种MyD88靶向的E3泛素连接酶。免疫共沉淀分析发现，TcpC能增强巨噬细胞MyD88的泛素化。体外泛素化检测实验结果显示，TcpC是一种能增强MyD88泛素化的E3连接酶。C12和W106是维持TcpC E3泛素链接酶活性的关键氨基酸。我们的研究在国内外首次发现，细菌来源的毒力因子可以作为MyD88靶向的E3泛素连接酶，通过促进MyD88泛素化降解，阻断TLR信号通路，阐明了TcpC介导的固有免疫逃逸新机制。