Vitamin D3 suppresses intestinal epithelial stemness via ER stress induction in intestinal organoids

Abstract

Abstract Background Vitamin D3 is important for normal function of the intestinal epithelial cells (IECs). In this study, we aimed to investigate the effects of vitamin D3 on the differentiation, stemness, and viability of healthy IECs in intestinal organoids. Methods Intestinal organoids derived from mouse small intestine were treated with vitamin D3, and the effects on intestinal stemness and differentiation were evaluated using real-time PCR and immunofluorescence staining of the distinct lineage markers. Cell viability was analyzed using viability and apoptosis assays. Results Vitamin D3 enhanced IEC differentiation into the distinct lineages of specialized IECs, including Paneth, goblet, and enteroendocrine cells and absorptive enterocytes. Decreased expression levels of leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) and the presence of several LGR5-green fluorescent protein (GFP)-positive cells were observed in vitamin D3-treated organoids derived from LGR5-GFP mice. The formation of the crypt-villus structure was also inhibited by vitamin D3, suggesting that vitamin D3 suppresses intestinal cell stemness. Furthermore, the expression levels of unfolded protein response genes, C/EBP homologous protein (CHOP), and activating transcription factor 6 (ATF6) were upregulated in vitamin D3-treated organoids. Moreover, vitamin D3 promoted apoptotic cell death in intestinal cells, which may be associated with the decrease in intestinal stemness. LGR5 gene expression, ISC number, and apoptotic cell death were partially recovered in the presence of the ER stress inhibitor tauroursodeoxycholic acid (TUDCA), suggesting that intestinal stemness suppression and intestinal apoptosis occurred via ER stress activation. Conclusions Our study provides important insights into the effects of vitamin D3 on the induction of IEC differentiation and apoptotic cell death, and inhibition of intestinal stemness accompanied by ER stress augmentation.

Keywords

• Intestinal epithelial cells (IECs)
• Intestinal organoids
• Vitamin D3
• Endoplasmic reticulum (ER) stress