title: Chromosome region maintenance-1 (CRM1) regulates apoptosis of intestinal epithelial cells via p27kip1 in Crohn's disease.
abstract: OBJECTIVE
  To investigate the role of chromosome region maintenance-1 (CRM1) in Crohn's disease (CD) and its potential pathological mechanisms.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  Up-regulated CRM1 may facilitate IEC apoptosis possibly through p27kip1 in CD, indicating an important role of CRM1 in the pathophysiology of CD.
authors.affiliation: Department of Gastroenterology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
authors.forename: Lijun
authors.initials: L
authors.lastname: Yan
authors.affiliation: Department of Gastroenterology, Affiliated Hospital of Nantong University, Nantong, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Gastroenterology, The Third Affiliated Hospital with Nanjing Medical University, Nanjing, China.
authors.forename: Jian'an
authors.initials: J
authors.lastname: Bai
authors.affiliation: Department of Gastroenterology, Affiliated Hospital of Nantong University, Nantong, China.
authors.forename: Xianjing
authors.initials: X
authors.lastname: Miao
authors.affiliation: Department of Gastroenterology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
authors.forename: Weiwen
authors.initials: W
authors.lastname: Zeng
authors.affiliation: Department of Gastroenterology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
authors.forename: Xiumei
authors.initials: X
authors.lastname: Hua
authors.affiliation: Department of Gastroenterology, Affiliated Hospital of Nantong University, Nantong, China.
authors.forename: Runzhou
authors.initials: R
authors.lastname: Ni
authors.affiliation: Department of Pathogen Biology, Medical College, Nantong University; Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, China.
authors.forename: Dongmei
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Department of Gastroenterology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China. Electronic address: tqy716219@126.com.
authors.forename: Qiyun
authors.initials: Q
authors.lastname: Tang
mesh_terms: D000818:Animals; D017209:Apoptosis; D003424:Crohn Disease / Q000473:pathology*; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000502:physiology*; D004847:Epithelial Cells; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology; D028884:Karyopherins / Q000502:physiology*; D051379:Mice; D018160:Receptors, Cytoplasmic and Nuclear / Q000502:physiology*; D000097606:Exportin 1 Protein
chemical_list: D050760:Cyclin-Dependent Kinase Inhibitor p27; D028884:Karyopherins; D018160:Receptors, Cytoplasmic and Nuclear; D000097606:Exportin 1 Protein; C493961:Cdkn1b protein, mouse