A65 HNF4A IS A KEY REGULATOR OF THE EPITHELIAL STEM CELL NICHE. (1st March 2018)

• Record Type: Journal Article
• Authors: Jones, C; Boudreau, F
• Is Part Of: Journal of the Canadian Association of Gastroenterology. Volume 1(2018)Supplement 2
• Page Start: 102
• Page End: 103
• Publication Date: 2018-03-01
• Subjects: Gastroenterology -- Periodicals; 616.33005
• DOI: 10.1093/jcag/gwy009.065
• Languages: English
• ISSNs: 2515-2084

Abstract

Abstract: Background: HNF4α is a transcriptional factor downregulated in intestinal bowel diseases (IBD). Epithelial deletion of HNF4α in the mouse intestine leads to spontaneous intestinal inflammation. Those observations suggest that HNF4α could play an important role in epithelial homeostasis. The intestinal crypt is the functional compartment responsible for the maintenance of this homeostasis. Proliferation of stem cells is essential for the mucosa healing following injuries, while Paneth cells are important regulators of this process through the secretion of stem cell niche factors including WNT3, a key activator of the canonical WNT/β-catenin pathway. Aims: We aimed to investigate if HNF4α could play an intrinsic role in stem and Paneth cells for the maintenance of the epithelial niche in intestinal crypts. Methods: Villin-Cre/HNF4α loxp/loxp and the hydroxytamoxifen (4OHT) inducible Villin-Cre ERT2/HNF4α loxp/loxp mouse models were used in this study. Isolated crypts were processed for protein and RNA isolation. Enteroids were derived from these models and used for RNAseq and qPCR experiments. Results: Enteroids were derived from jejunal crypts of inducible Villin-Cre ERT2/HNF4α loxp/loxp mice. Induction of HNF4α deletion with 4OHT led to degeneration of these enteroids starting 5 days after the deletion, an observation reminiscent of enteroids derived from the Villin-Cre/HNF4α loxp/loxp mouse model. EdU incorporation assays showed a decrease in the proliferative rate of enteroids 4 days following HNF4α deletion. RNAseq was next performed on RNA isolated from enteroids induced for HNF4α deletion after 2 and 4 days in culture. Transcriptomic analysis identified more than a thousand of genes differentially expressed following the deletion of HNF4α under these conditions. A significant reduction of WNT3 was predicted, an observation that was further confirmed by qPCR and Western in jejunal crypts of HNF4α mutant mice. To measure the functional relevance of WNT3 reduction during enteroids degeneration, a rescue experiment was performed. WNT3A supplementation was able to maintain HNF4α deleted enteroids in culture. Transcriptomic analysis of WNT3A-treated enteroids showed rescue for 85% of the genes identified to be modulated in enteroids deleted for HNF4α. To further verify if HNF4α may contribute to Paneth cell differentiation, enteroids committed to differentiate into the Paneth lineage were deleted for HNF4α. Gene transcript expression of Paneth cell markers (Defa3, Defa5, Defa20, Defa21-22, Lyz and WNT3) were all downregulated in the absence of HNF4α as opposed to EpHB3, which was not significantly modulated. Conclusions: This study identifies HNF4α as a key regulator of Paneth cell function for maintenance of the epithelial stem cell niche. These observations provide a novel mechanistic loop for which the intestinal epithelial healing process could be dependent following stress-related injuries. Funding Agencies: CIHRCCC-Vertex