Chromosome region maintenance-1 (CRM1) regulates apoptosis of intestinal epithelial cells via p27kip1 in Crohn's disease

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.