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ROLE OF CUX1 DURING INTESTINAL EPITHELIAL CELL RESTITUTION FOLLOWING INJURY
R Latreille, M Darsigny, F BoudreauUniversité de Sherbrooke, Sherbrooke, QC
Aims: Our laboratory has recently shown that mutant mice for the transcriptional regulator Cux1 can initiate a severe intestinal inflammatory response as compared to control mice. One characteristic associated with the phenotype of these mutant mice was the inability to renew their intestinal epithelium following the induction of a chemical stress with dextran sodium sulfate (DSS). We hypothesize that the transcription factor Cux1 could be functionally involved during the process of epithelial restitution in the context of inflammatory bowel diseases.
Methods: Rat intestinal epithelial cells (IEC-6) were stably infected with lentivirus/shRNA in order to downmodulate the expression of Cux1. Wounding assays were performed by scratching IEC-6 confluent cells with a razor blade to provide migration of cells in the injury. TGFb was supplemented into petri dishes to stop proliferation and promote cell migration. Quantitative PCR and Western bloting analysis were performed to identify signalling targets of Cux1 under TGFb dependent stimulation. These targets were also studied in a hypomorphic mouse model for Cux1 treated with DSS to induce intestinal inflammatory disorders.
Results: Inducing an injury on Cux1 deficient IEC-6 cells resulted in their inability to migrate as compared to control cells. Supplementation of TGFb under these conditions led to a reduction of cell proliferation as determined by BrdU incorporation and immunofluorescence assays. However, TGFb promoting effect on cell migration was significantly impaired in IEC-6 cells that harboured reduced Cux1 expression. Real-time PCR and Western blot analysis identified the TGFb associated kinase 1 (TAK1) to be downmodulated in the absence of Cux1. In addition, IRAK4, a crucial kinase involved in Toll-like receptor signalling, was reduced more than 40% in Cux1 deficient IEC-6 cells. This tendency was also observed at the level of the gene transcript in the intestine of Cux1 mutant mice.
Conclusions: Our results highlight an important role of the transcription factor Cux1 during the restitution process of the intestinal epithelium. The nature of Cux1 functional interaction with TAK1 and IRAK4 in a context of inflammatory bowel diseases is currently under investigation. Supported by the CCFC.