肠上皮细胞特异性OTUB1在DSS诱导的结肠炎中的功能及分子机制研究

Intestinal epithelial cells (IECs) play critical roles in inflammatory bowel disease (IBD) and its animal model DSS-induced colitis (DIC). OTUB1, a K48-specific deubiquitinating enzyme (DUB), has been shown to regulate multiple signaling pathways including the NF-κB pathway. Functionally, OTUB1 plays key roles in various diseases such as autoimmune diseases, Alzheimer’s disease, and cancer, but its function in DIC and IBD is still unknown. In our preliminary study, we found that protein levels of OTUB1 were upregulated in IECs during DIC. To study the in vivo function of OTUB1, we generated Villin-CreERT2 OTUB1fl/fl mice, in which OTUB1 could be ablated specifically in IECs upon treatment with tamoxifen. In comparison to OTUB1fl/fl control mice, Villin-CreERT2 OTUB1fl/fl mice were more susceptible to DSS-induced acute and chronic colitis, suggesting that IEC-specific OTUB1 is protective in this disease model. In the proposed project, we will apply a broad range of techniques, such as RNA sequencing, proteomics, and construction of OTUB1 mutant mice, to elucidate the functional role of IEC-specific OTUB1 in DIC as well as the mechanism of action of OTUB1 in signal transduction, thereby providing potential novel targets for the treatment of IBD.

肠上皮细胞（IEC）在人类炎症性肠病（IBD）和其动物模型DSS诱导的结肠炎（DIC）中发挥多种关键性的作用。去泛素化酶OTUB1可以调控包括NF-κB在内的多种信号通路，在自发免疫性疾病、神经退行性疾病和癌症等疾病中发挥重要的作用。然而，OTUB1在肠炎中的作用尚不明确。我们前期的研究发现，IEC中的OTUB1在DIC中表达上调。为了研究OTUB1的体内功能，我们构建了Villin-CreERT2 OTUB1fl/fl小鼠，该小鼠IEC中的OTUB1可以受他莫昔芬诱导敲除。与对照组小鼠相比，Villin-CreERT2 OTUB1fl/fl小鼠对DIC更加敏感，提示OTUB1具有缓解结肠炎的作用。在本项目中，我们拟通过转录组测序、蛋白质组学和构建OTUB1突变体小鼠等技术，阐明IEC特异性OTUB1在DIC中的功能及其调控信号转导的分子机制，为IBD的防治提供潜在的新靶点。

肠上皮细胞（IEC）不仅作为物理屏障来隔离肠道微生物，还可以协助免疫系统识别肠道抗原，在人类炎症性肠病（IBD）和其动物模型DSS诱导的结肠炎中发挥关键性作用。去泛素化酶OTUB1可以调控多种炎症信号通路，在中枢神经系统自身免疫性疾病和神经退行性疾病等炎症性疾病中发挥重要的作用。然而，OTUB1在结肠炎中的作用尚不明确。在本项目中，我们发现小鼠IEC中的OTUB1在结肠炎中表达上调，提示IEC中的OTUB1在结肠炎中可能发挥关键的调控作用。为了研究IEC特异性OTUB1在结肠炎中的功能，我们构建了他莫昔芬诱导型IEC细胞特异性敲除（Villin-CreERT2 OTUB1fl/fl）小鼠。在DSS诱导的急性和慢性结肠炎中，Villin-CreERT2 OTUB1fl/fl小鼠发展出比对照组（OTUB1fl/fl）小鼠更严重的结肠炎。在体外，受到TNF和LPS刺激时，敲除OTUB1的MODE-K肠上皮细胞系表现出更显著的NF-κB信号活化且产生更多的炎症因子。我们发现组蛋白甲基转移酶EZH2为OTUB1直接调控的蛋白靶点，而前期报道显示IEC来源的EZH2可以抑制DSS诱导的结肠炎。敲除OTUB1可以显著降低MODE-K细胞中EZH2的蛋白丰度，却不影响EZH2的转录。在机制上，OTUB1通过抑制泛素-蛋白酶体介导的EZH2降解而增加其细胞内的蛋白水平。总之，本项目的实施发现了新型IBD调控机制，并为IBD的防治鉴定出一个潜在靶点。