Acute exposure to deoxynivalenol inhibits porcine enteroid activity via suppression of the Wnt/ß-catenin pathway.

The intake of food containing deoxynivalenol frequently causes damage to the intestine, the renewal of which is driven by intestinal stem cells (ISCs). Nevertheless, the toxicity of deoxynivalenol on ISCs and its underlying mechanisms remain to be elucidated. As pigs are the most sensitive animals to deoxynivalenol, we used piglets for investigation in this study. Here, we show that intestinal epithelial cell activity, B cell-specific Moloney murine leukemia virus insertion site 1 (Bmi1) protein level, and Wnt/ß-catenin pathway activity were suppressed with acute expose to deoxynivalenol. We further established a novel system for porcine crypt isolation and ex vivo cultivation. Crypts and crypt cells expanded and budded with typical enteroid morphologies under this system. Our results show that both acute in vivo and in vitro administration of deoxynivalenol significantly decreased enteroid activity. Simultaneously, protein levels of ß-catenin and leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5) in enteroids were reduced by deoxynivalenol exposure. In conclusion, we established a reliable culture system for porcine enteroids and demonstrated for the first time that the activity of ISCs and the Wnt/ß-catenin pathway is sensitively suppressed by acute deoxynivalenol exposure.

CDX2 Stimulates the Proliferation of Porcine Intestinal Epithelial Cells by Activating the mTORC1 and Wnt/ß-Catenin Signaling Pathways.

Caudal type homeobox 2 (CDX2) is expressed in intestinal epithelial cells and plays a role in gut development and homeostasis by regulating cell proliferation. However, whether CDX2 cooperates with the mammalian target of rapamycin complex 1 (mTORC1) and Wnt/ß-catenin signaling pathways to stimulate cell proliferation remains unknown. The objective of this study was to investigate the effect of CDX2 on the proliferation of porcine jejunum epithelial cells (IPEC-J2) and the correlation between CDX2, the mTORC1 and Wnt/ß-catenin signaling pathways. CDX2 overexpression and knockdown cell culture models were established to explore the regulation of CDX2 on both pathways. Pathway-specific antagonists were used to verify the effects. The results showed that CDX2 overexpression increased IPEC-J2 cell proliferation and activated both the mTORC1 and Wnt/ß-catenin pathways, and that CDX2 knockdown decreased cell proliferation and inhibited both pathways. Furthermore, the mTORC1 and Wnt/ß-catenin pathway-specific antagonist rapamycin and XAV939 (3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)]-4H -thiopyrano[4,3-d]pyrimidin-4-one) both suppressed the proliferation of IPEC-J2 cells overexpressing CDX2, and that the combination of rapamycin and XAV939 had an additive effect. Regardless of whether the cells were treated with rapamycin or XAV939 alone or in combination, both mTORC1 and Wnt/ß-catenin pathways were down-regulated, accompanied by a decrease in CDX2 expression. Taken together, our data indicate that CDX2 stimulates porcine intestinal epithelial cell proliferation by activating the mTORC1 and Wnt/ß-catenin signaling pathways.

CDX2 increases SLC7A7 expression and proliferation of pig intestinal epithelial cells.

Nutrient absorption mediated by nutrient transporters expressed in the intestinal epithelium supplies substrates to support intestinal processes, including epithelial cell proliferation. We evaluated the role of Caudal type homeobox 2 (CDX2), an intestine-specific transcription factor, in the proliferation of pig intestinal epithelial cells (IPEC-1) and searched for novel intestinal nutrient transporter genes activated by CDX2. Our cloned pig CDX2 cDNA contains a "homeobox" DNA binding motif, suggesting it is a transcriptional activator. CDX2 overexpression in IPEC-1 cells increased cell proliferation, the percentage of cells in S/G2 phase, and the abundance of transcripts of the cell cycle-related genes Cyclin A2; Cyclin B; Cyclin D2; proliferating cell nuclear antigen; and cell cycle cyclin-dependent kinases 1, 2 and 4, as well as the predicted CDX2 target genes SLC1A1, SLC5A1 and SLC7A7. In addition, luciferase reporter and chromatin immunoprecipitation assays revealed that CDX2 binds directly to the SLC7A7 promoter. This is the first report of CDX2 function in pig intestinal epithelial cells and identifies SLC7A7 as a novel CDX2 target gene. Our findings show that nutrient transporters are activated during CDX2-induced proliferation of normal intestinal epithelial cells.