Yan Lijun, Wang Liang, Bai Jian'an, Miao Xianjing, Zeng Weiwen, Hua Xiumei, Ni Runzhou, Zhang Dongmei, Tang Qiyun
Department of Gastroenterology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Department of Gastroenterology, Affiliated Hospital of Nantong University, Nantong, China.
Clin Res Hepatol Gastroenterol. 2017 Sep;41(4):445-458. doi: 10.1016/j.clinre.2017.01.012. Epub 2017 Mar 9.
To investigate the role of chromosome region maintenance-1 (CRM1) in Crohn's disease (CD) and its potential pathological mechanisms.
The expression and distribution of CRM1 in mucosal biopsies from patients with active CD and normal controls were detected by immunohistochemistry (IHC). We established a murine model of acute colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). Western blot was performed to investigate the expression levels of CRM1, apoptotic markers (active caspase-3 and cleaved PARP), p27kip1 and p-p27ser10. IHC was performed to evaluate the distribution of CRM1, and double immunofluorescence (IF) was performed to evaluate the co-localization of CRM1 and active capase-3. Cells of the human intestinal epithelial cell line HT-29 were incubated with tumor necrosis factor-α (TNF-α) to establish an apoptotic in vitro model. Western blot was performed to determine the expression levels of CRM1, active caspase-3, cleaved PARP and p-p27ser10. Cytoplasmic and nuclear extracts were assessed to examine the translocation of CRM1. The interaction between CRM1 and p27kip1 was assessed by co-immunoprecipitation (co-IP) assays. Furthermore, we used small interfering RNA (siRNA) to knock down the protein expression of CRM1 in HT-29 cells and then measured the expression of active caspase-3, cleaved PARP and p-p27ser10. Flow cytometry was used to determine the effect of CRM1 on intestinal epithelial cell (IEC) apoptosis.
We observed up-regulation of CRM1 accompanied by elevated levels of IEC apoptotic markers (active caspase-3 and cleaved PARP) and p-p27ser10 in IECs of patients with active CD and in TNBS-induced colitis model cells. However, the expression of p27kip1 was negatively correlated with the expression patterns of CRM1, p-p27ser10 and apoptotic biochemical markers. Co-localization of CRM1 and active caspase-3 in IECs of the TNBS group further indicated the possible involvement of CRM1 in IEC apoptosis. By employing TNF-α-treated HT-29 cells as an in vitro IEC apoptosis model, we found that the expression levels of CRM1 and p-p27ser10 were in accordance with active caspase-3 and cleaved PARP. In addition, immunoprecipitation confirmed the physical interaction between CRM1 and p27kip1. siRNA knockdown of CRM1 significantly inhibited the phosphorylation of p27kip1 and the expression of active caspase-3 and cleaved PARP. In addition, flow cytometry analysis also showed that silencing CRM1 by siRNA inhibited TNF-α-induced cellular apoptosis in HT-29 cells.
Up-regulated CRM1 may facilitate IEC apoptosis possibly through p27kip1 in CD, indicating an important role of CRM1 in the pathophysiology of CD.
探讨染色体区域维持蛋白1（CRM1）在克罗恩病（CD）中的作用及其潜在病理机制。
采用免疫组织化学（IHC）检测活动期CD患者及正常对照者黏膜活检组织中CRM1的表达及分布。我们建立了2,4,6-三硝基苯磺酸（TNBS）诱导的急性结肠炎小鼠模型。采用蛋白质免疫印迹法检测CRM1、凋亡标志物（活化的半胱天冬酶-3和裂解的PARP）、p27kip1和p-p27ser10的表达水平。采用IHC评估CRM1的分布，采用双重免疫荧光（IF）评估CRM1与活化的半胱天冬酶-3的共定位。将人肠上皮细胞系HT-29细胞与肿瘤坏死因子-α（TNF-α）孵育以建立体外凋亡模型。采用蛋白质免疫印迹法测定CRM1、活化的半胱天冬酶-3、裂解的PARP和p-p27ser10的表达水平。评估细胞质和细胞核提取物以检测CRM1的转位。通过免疫共沉淀（co-IP）实验评估CRM1与p27kip1之间的相互作用。此外，我们使用小干扰RNA（siRNA）敲低HT-29细胞中CRM1的蛋白表达，然后检测活化的半胱天冬酶-3、裂解的PARP和p-p27ser10的表达。采用流式细胞术测定CRM1对肠上皮细胞（IEC）凋亡的影响。
我们观察到活动期CD患者的IECs以及TNBS诱导的结肠炎模型细胞中CRM1上调，同时IEC凋亡标志物（活化的半胱天冬酶-3和裂解的PARP）和p-p27ser10水平升高。然而，p27kip1的表达与CRM1、p-p27ser10和凋亡生化标志物的表达模式呈负相关。TNBS组IECs中CRM1与活化的半胱天冬酶-3的共定位进一步表明CRM1可能参与IEC凋亡。通过将TNF-α处理的HT-29细胞作为体外IEC凋亡模型，我们发现CRM1和p-p27ser10的表达水平与活化的半胱天冬酶-3和裂解的PARP一致。此外，免疫沉淀证实了CRM1与p27kip1之间存在物理相互作用。siRNA敲低CRM1显著抑制p27kip1的磷酸化以及活化的半胱天冬酶-3和裂解的PARP的表达。此外，流式细胞术分析还显示，通过siRNA沉默CRM1可抑制TNF-α诱导的HT-29细胞凋亡。
CD中上调的CRM1可能通过p27kip1促进IEC凋亡，表明CRM1在CD病理生理学中起重要作用。
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