Abstract：
OBJECTIVE: Inflammatory bowel disease (IBD) is currently gaining an increasing global interest. Intestinal epithelial barrier dysfunction is crucial toward developing IBD; however, the underlying mechanisms are not yet elucidated. This study is aimed at elucidating the function of CRL4DCAF2, an E3 ligase, toward mediating intestinal homeostasis.
METHODS: Colon samples were collected from patients with IBD and healthy individuals to examine the expression of CRL4DCAF2. CRL4DCAF2 conditional knockdown in mouse intestinal epithelial cells (IECs) (DCAF2EKD) were constructed. DCAF2EKD and their littermate control (DCAF2EWT) were treated with dextran sodium sulfate (DSS) to induce acute colitis. Transcriptome analysis was performed on inflamed colon samples obtained from the mice. Cell cycle regulators were evaluated using real-time polymerase chain reaction (PCR), while tight junction and apoptosis proteins were examined via immunofluorescence and western blot.
RESULTS: CRL4DCAF2 expression was significantly decreased in the inflamed IBD epithelium, and low expression of CRL4DCAF2 associated with high recurrence risk. Mice with DCAF2 specific knockout in IECs suffer from embryonic death. Multiple genes involved in cell proliferation, immune response, and gap junction were differentially expressed in inflamed colon from DCAF2EKD compared with DCAF2EWT. Furthermore, conditional downregulation of CRL4DCAF2 in the intestinal epithelium induced primarily epithelial damage, increased intestinal permeability, and diminished tight junction protein expression. In vivo and in vitro cell transfection experiments revealed that CRL4DCAF2 enhanced cell proliferation by promoting p21 ubiquitination and degradation, thereby inhibiting G2/M cell cycle. In addition, CRL4DCAF2 can also inhibit IEC apoptosis and promote cell autophagy.
CONCLUSIONS: CRL4DCAF2 downregulation in IECs promotes intestinal barrier dysfunction and inhibits IEC proliferation, thus making it more susceptible to inflammation.
METHODS: Colon samples were collected from patients with IBD and healthy individuals to examine the expression of CRL4DCAF2. CRL4DCAF2 conditional knockdown in mouse intestinal epithelial cells (IECs) (DCAF2EKD) were constructed. DCAF2EKD and their littermate control (DCAF2EWT) were treated with dextran sodium sulfate (DSS) to induce acute colitis. Transcriptome analysis was performed on inflamed colon samples obtained from the mice. Cell cycle regulators were evaluated using real-time polymerase chain reaction (PCR), while tight junction and apoptosis proteins were examined via immunofluorescence and western blot.
RESULTS: CRL4DCAF2 expression was significantly decreased in the inflamed IBD epithelium, and low expression of CRL4DCAF2 associated with high recurrence risk. Mice with DCAF2 specific knockout in IECs suffer from embryonic death. Multiple genes involved in cell proliferation, immune response, and gap junction were differentially expressed in inflamed colon from DCAF2EKD compared with DCAF2EWT. Furthermore, conditional downregulation of CRL4DCAF2 in the intestinal epithelium induced primarily epithelial damage, increased intestinal permeability, and diminished tight junction protein expression. In vivo and in vitro cell transfection experiments revealed that CRL4DCAF2 enhanced cell proliferation by promoting p21 ubiquitination and degradation, thereby inhibiting G2/M cell cycle. In addition, CRL4DCAF2 can also inhibit IEC apoptosis and promote cell autophagy.
CONCLUSIONS: CRL4DCAF2 downregulation in IECs promotes intestinal barrier dysfunction and inhibits IEC proliferation, thus making it more susceptible to inflammation.
摘要：
目的：炎症性肠病（IBD）目前正在引起全球越来越多的关注。肠上皮屏障功能障碍对IBD的发展至关重要；然而，潜在的机制尚未阐明。本研究旨在阐明E3连接酶CRL4DCAF2的功能,向介导肠道稳态。
方法：收集IBD患者和健康个体的结肠样本，检测CRL4DCAF2的表达。构建小鼠肠上皮细胞(IECs)中的CRL4DCAF2条件性敲除(DCAF2EKD)。用葡聚糖硫酸钠（DSS）处理DCAF2EKD及其同窝对照（DCAF2EWT）以诱导急性结肠炎。对得自小鼠的发炎结肠样品进行转录组分析。使用实时聚合酶链反应（PCR）评估细胞周期调节剂，同时通过免疫荧光和蛋白质印迹检测紧密连接和凋亡蛋白。
结果：炎症IBD上皮中CRL4DCAF2表达显著降低，CRL4DCAF2低表达与高复发风险有关。在IECs中具有DCAF2特异性敲除的小鼠遭受胚胎死亡。参与细胞增殖的多个基因，免疫反应，与DCAF2EWT相比，DCAF2EKD在发炎的结肠中差异表达。此外，CRL4DCAF2在肠上皮中的条件性下调主要诱导上皮损伤，肠道通透性增加，和减少紧密连接蛋白的表达。体内和体外细胞转染实验表明，CRL4DCAF2通过促进p21泛素化和降解来增强细胞增殖，从而抑制G2/M细胞周期。此外,CRL4DCAF2还可以抑制IEC凋亡和促进细胞自噬。
结论：IECs中CRL4DCAF2下调促进肠屏障功能障碍并抑制IEC增殖，从而使其更容易发炎。
方法：收集IBD患者和健康个体的结肠样本，检测CRL4DCAF2的表达。构建小鼠肠上皮细胞(IECs)中的CRL4DCAF2条件性敲除(DCAF2EKD)。用葡聚糖硫酸钠（DSS）处理DCAF2EKD及其同窝对照（DCAF2EWT）以诱导急性结肠炎。对得自小鼠的发炎结肠样品进行转录组分析。使用实时聚合酶链反应（PCR）评估细胞周期调节剂，同时通过免疫荧光和蛋白质印迹检测紧密连接和凋亡蛋白。
结果：炎症IBD上皮中CRL4DCAF2表达显著降低，CRL4DCAF2低表达与高复发风险有关。在IECs中具有DCAF2特异性敲除的小鼠遭受胚胎死亡。参与细胞增殖的多个基因，免疫反应，与DCAF2EWT相比，DCAF2EKD在发炎的结肠中差异表达。此外，CRL4DCAF2在肠上皮中的条件性下调主要诱导上皮损伤，肠道通透性增加，和减少紧密连接蛋白的表达。体内和体外细胞转染实验表明，CRL4DCAF2通过促进p21泛素化和降解来增强细胞增殖，从而抑制G2/M细胞周期。此外,CRL4DCAF2还可以抑制IEC凋亡和促进细胞自噬。
结论：IECs中CRL4DCAF2下调促进肠屏障功能障碍并抑制IEC增殖，从而使其更容易发炎。
