DOP002. Conditional deletion of Prep1 in the intestinal epithelium alters epithelial homeostasis, intestinal development, and controls colitis susceptibility
S. D'Alessio1, C. Tacconi1, A. Gandelli1, C. Correale1, M. Genua1, S. Vetrano1, F. Blasi2, S. Danese1, 1Humanitas Clinical and Research Center, IBD Center, Rozzano, Italy, 2IFOM-IEO Campus, Transcriptional mechanisms in development and tumor formation, Milan, Italy
Balanced and dynamic interactions among mucus layers, intestinal epithelial cells, and microbiota, are essential for the maintenance of the intestinal mucosal homeostasis. The disruption of this balance leads to a defective mucus barrier with increased permeability that results in intestinal inflammation. The homeodomain transcription factor, Prep1, is expressed in the post-mitotic differentiated intestinal epithelial cells and is essential in embryonic development. Here we report for the first time the involvement of Prep1 in intestinal epithelial homeostasis, and its functional role in experimental colitis.
Mice lacking Prep1 in intestinal epithelium were generated by mating Prep1flox/flox mice with VillinCRE animals. Goblet cells staining was carried out by Alcian Blue and Periodic Acid-Schiff (PAS) staining, while mucins were quantified by real-time PCR and immunohistochemistry. The dextran sulfate sodium (DSS) model of colitis was used in VillinCRE/Prep1flox/flox and wild-type (wt) mice. Moreover, Prep1 expression was evaluated in the intestinal epithelium of surgical specimens from normal individuals and patients with IBD, by Western Blot. Cross-linked, sonicated chromatin from human intestinal epithelial cells, was then immunoprecipitated with an anti-Prep1 antibody and the precipitated DNA analyzed by real-time PCR with a set of primers within the MUC1, MUC2, and MUC13 promoters (ChiP).
Mice with conditional ablation of the Prep1 gene from the intestinal epithelium were viable. However, transgenic animals appeared smaller than wt, with a defective development of both the small and large intestine. VillinCRE/Prep1flox/flox mice showed an altered Goblet cell maturation and differentiation, resulting in MUC2 (soluble mucin) overproduction and MUC1/MUC13 (membrane-bound mucins) overexpression. Mutant mice were protected from DSS-induced colitis, as assessed by body weight loss, colitis activity Index, and histological scores. This correlated with reduced intestinal permeability (p < 0.001), and bacterial penetration (p < 0.05) within the inner mucus layer. Finally, Prep1 was find to be up-regulated in the intestinal epithelium of IBD patients, when compared to controls, and ChiP assay revealed that Prep1 modulate the transcription of the human MUC2, MUC1 and MUC13 genes, by directly binding to their promoters and acting as a repressor.
Our findings demonstrate an essential role for Prep1 in intestinal development, epithelial homeostasis and maintenance of the mucosal barrier. Its manipulation, could indeed represent a novel therapeutic approach for the treatment of IBD, possibly restoring the mucin barrier.