Tu1733 MicroRNA-322/503 Repress Translation of Smurf1 and Smurf2 to Regulate TGFβ/SMAD Signaling and Gut Epithelial Homeostasis

Background and aims: The intestinal epithelium is a vigorously self-renewing tissue. Stem cells and transient-amplifying cells are located at the base of the crypts and give rise to terminally differentiated cells which migrate up the villous tip. A tightly regulated balance between proliferation and cell death is of utmost importance. Survivin is a unique member of the inhibitor of apoptosis (IAP) family with bifunctional properties involved in controlling apoptosis and cell division, respectively. To elucidate the contribution of survivin in the intestine we analysed mice with a specific deletion of survivin in intestinal epithelial cells (IECs). Methods: Survivinfl mice were crossbred to Villin-Cre or Villin-CreERT2 mice, resulting in conditional knockout mice (SurvivinΔIEC) or inducible knockout mice (SurviviniΔIEC). SurviviniΔIEC and control mice were histologically investigated by immuno¬histochemistry. The gene and protein expression pattern of IECs of Survivini ΔIEC mice was analysed by quantitative PCR and Western-Blot. To analyse the timepoint of the survivin deletion and intestinal epithelial cell death we focused on the in-vitro cultivation of organoids from SurviviniΔIEC mice. Results: We initially intended to generate mice with a specific deletion of the survivin gene in IECs but mice were embryonic lethal. Therefore, we created mice with an inducible deletion of the survivin gene in IECs, triggered by a daily injection of tamoxifen. Immunohistochemical staining of control gut tissue revealed a localisation of survivin in the crypt compartment. Detailed analysis showed specific presence of survivin in transient amplifying cells and stem cells. The specific deletion of survivin in IECs leads to a fatal destruction of the intestinal epithelium. IECs of Survivini ΔIEC mice showed abnormally enlarged nuclei, suggesting a defect in cell cycle progression. Furthermore, we observed accumulations of encapsulated debri and a massive dislocation of the crypt compartment. Western-blot analysis revealed elevated protein levels of activated caspase-3 and caspase-8, indicating dysregulated cell death of survivin depleted IECs. Quantitative PCR showed reduced gene expression levels of stem cell genes like lgr5 and olfm4, as well as for the proliferation marker ki67. Conclusions: The dramatic phenotype of Survivini ΔIEC mice demonstrated the absolute necessity of survivin in the intestinal epithelium. The lack of survivin results in a complete loss of the crypt-villous structure and the prominent formation of enlarged nuclei, arrested in cell cycle. The loss of stem cells as well as transient amplifying cells demonstrates the important contribution of survivin for the intestinal stem cell niche and the preservation of epithelial cells in the gut.