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

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. 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. 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. 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.

Background 1,25-Dihydroxyvitamin D 3 (vitamin D 3 ) is an active form of vitamin D [1,2]. Vitamin D 3 has a broad range of biological activities and is primarily responsible for intestinal absorption of calcium and phosphorus [3,4]. In addition, vitamin D 3 plays a role in maintaining intestinal barrier function, which prevents bacterial translocation and ensures that appropriate inflammatory responses take place by regulating tight junction gene expression in the intestine [5,6]. Furthermore, vitamin D 3 contributes to detoxification, protection against infection, and cancer suppression [7,8]. Vitamin D 3 deficiency is associated with many intestinal diseases, such as inflammatory bowel diseases, short bowel syndrome, and pancreatitis [8,9]. Moreover, supplementation with high-dose vitamin D 3 was found to increase colitis susceptibility in a dextran sulfate sodium-induced colitis mouse model [10]. However, direct evidence showing the effect of vitamin D 3 on intestinal epithelial cells (IECs) in the small intestine under homeostasis is limited. Therefore, it is necessary to study the dosedependent effects of vitamin D 3 on IECs in the small intestine.
IECs form a single layer epithelium, which functions as a physical barrier and supports host health [11]. IECs comprise various specific cell lineages originating from intestinal stem cells (ISCs). ISCs undergo proliferation and differentiation into secretory cells, including Paneth (lysozyme-producing cells), goblet (mucin-producing cells), and enteroendocrine cells, as well as absorptive enterocytes [12]. As vitamin D 3 is easily absorbed by IECs in the small intestine, many studies have focused on the effect of vitamin D 3 on IEC function [13][14][15]. The importance of vitamin D 3 on IEC function has been proven using in vitro or in vivo studies. For example, vitamin D 3 is known to protect against colorectal cancer by suppressing epithelial cell proliferation and inducing apoptosis [16,17], while increased apoptotic cell death was observed in the small intestine and colon of vitamin D receptor (VDR)-deficient mice [18]. Furthermore, vitamin D 3 was found to promote the differentiation of colon carcinoma cells [19]. Indeed, consumption of low levels of dietary vitamin D and calcium in a semipurified diet or VDR inactivation in leucine-rich repeatcontaining G-protein-coupled receptor 5 (Lgr5)-positive ISCs leads to dysfunction of Lgr5 + ISCs [20], suggesting that vitamin D 3 is important for the function of IECs, especially that of ISCs.
Endoplasmic reticulum (ER) stress occurs under homeostatic conditions in the small intestine and is enhanced in intestinal diseases [21]. A variety of stimuli can induce ER stress, including infections, loss of cellular calcium homeostasis, and accumulation of unfolded or misfolded proteins [22]. ER stress subsequently activates the unfolded protein response (UPR), which is necessary for the restoration of normal cell functions, including those of IECs. In general, UPR involves either the survival or apoptotic pathway depending on the ER stress severity [23,24]. Indeed, UPR is also known to be important for the functions of goblet and Paneth cells, as well as the differentiation of ISCs into transit-amplifying (TA) cells [25,26]. Therefore, ER stress activation might mediate the effect of vitamin D 3 on IECs.
Intestinal organoids can mimic ISC proliferation and differentiation forming an intestinal crypt-villus-like structure [27]. Therefore, we utilized intestinal organoid cultures to study the dose-dependent effects of vitamin D 3 on IEC viability and function, especially in terms of stemness, differentiation, and survival. In addition, we aimed to examine signaling pathways implicated in vitamin D 3 action.

Preparation of intestinal organoids derived from mouse small intestine
The small intestine was dissected from male C57BL/6N mice (ORIENT Bio, Korea) or LGR-EGFP-IRES-CreERT2 (LGR5-GFP) mice, which were kindly provided by Professor Mi-Na Kweon (College of Medicine/Asan Medical Center, Korea). Intestinal crypts were isolated using the Gentle Cell Dissociation Reagent (StemCell Technologies, MA). The tissue was incubated with 0.1% bovine serum albumin (BSA), and the cell suspension was passed through a 70-μm cell strainer. The isolated crypts were observed under a microscope (CKX53, OLYMPUS, Japan). The crypts were mixed with Matrigel (BD Biosciences, NJ) and Intesticult TM OGM Mouse Basal Medium (StemCell Technologies) at a ratio of 1:1, and 20 μl of the suspended crypts was plated in 48-well plates. After polymerization by incubating at 37°C for 20 min, 400 μl of Intesticult TM OGM Mouse Basal Medium was added, and the plate was placed in a humidified incubator (5% CO 2 ) at 37°C. The culture medium was replaced every 2 days, and the organoids were passaged every 5 to 6 days.
For passaging, the culture medium was removed, and the organoids were recovered from the Matrigel using the Cell Recovery Solution (Corning, NY). After mechanical disruption by pipetting, the suspended crypts were transferred to microtubes and centrifuged at 850×g for 5 min. The crypt pellets were mixed with Matrigel and Intesticult TM OGM Mouse Basal Medium and cultured.

Vitamin D 3 treatment
Vitamin D 3 (1,25-dihydroxyvitamin D 3 ) was purchased from Sigma (Sigma-Aldrich, MO) and prepared following the manufacturer's instructions. Ethanol was used as a vehicle control. Intestinal organoids were treated with various concentrations of vitamin D 3 (10, 50, and 100 nM), and biological changes were observed after 3 days.

Endoplasmic reticulum (ER) stress inhibition
Tauroursodeoxycholic acid (TUDCA, MO) was used as a broad ER stress inhibitor. TUDCA was dissolved in dimethyl-sulphoxide (DMSO) according to the manufacturer's protocol. Intestinal organoids were treated with TUDCA at a concentration of 250 or 500 μM in the presence of vitamin D 3 .

Assessment of organoid budding
Following treatment with vitamin D 3 for 3 days, the morphology of intestinal organoids was observed under a light microscope, and the budding was analyzed by measuring the area between expanded organoids from the core using the ImageJ software. The relative value was compared with the non-treated group and presented as the percentage of budding organoids. Data were obtained from four to ten randomly selected fields to acquire ten individual organoids for budding assessment.

Assessment of organoid viability using MTT reduction
Organoid viability was evaluated using a cell proliferation kit (Roche, Germany). In brief, after culture medium removal, 10 μl MTT-labeling reagent was added to the organoid culture for 1 h. Viable organoids, which could reduce the MTT reagent to formazan, were imaged using a light microscope. After adding DMSO to solubilize the formazan crystals, the absorbance of the colored solution was measured using a microplate reader at 575 nm and a reference at 650 nm. Cell viability was calculated as the percentage of viable cells relative to the non-treated group.

Detection of apoptosis using the TUNEL assay
The organoids were fixed with 4% paraformaldehyde, and apoptosis was detected using the TUNEL assay kit-BrdU-Red (Abcam, MA) according to the manufacturer's protocol. Briefly, the fixed organoids were incubated with 70% ethanol for 30 min and then incubated with the DNA-labeling and antibody solutions for 60 and 30 min, respectively. Then, the organoids were stained with DAPI (Sigma-Aldrich) for 1 h and imaged using a confocal microscope (LSM 710; Carl Zeiss) at the Soonchunhyang Biomedical Research Core Facility of the Korea Basic Science Institute.

Immunofluorescence staining
After medium removal, the organoids were fixed with 4% paraformaldehyde. Permeabilization was performed with 0.2% Triton X-100 followed by a blocking step using 5% BSA. The organoids were incubated with the following primary antibodies: anti-Lgr5 (Abgent, CA), anti-Ki67 (Cell Signaling Technology, MA), anti-Lysozyme (Diagnostic Biosystems, CA), anti-Mucin 2 (Santa Cruz Biotechnology, CA), anti-Chromogranin A (Santa Cruz Biotechnology), anti-Villin (Santa Cruz Biotechnology), and anti-cleaved caspase-3 (Cell Signaling Technology) at 4°C overnight. Then, the organoids were incubated with the secondary antibodies, either Alexa Fluor 488-conjugated anti-mouse IgG (Life Technologies, MD) or Alexa Fluor 555-conjugated anti-rabbit IgG (Life Technologies), at room temperature for 2 h. The nuclei were stained with DAPI (Sigma-Aldrich) for 1 h, and the organoids were imaged using a confocal microscope (LSM 710; Carl Zeiss). The mean fluorescence intensity was analyzed with the ImageJ software, and the intensity of each marker was normalized to that of DAPI.

Quantitative real-time polymerase chain reaction (qPCR)
The intestinal organoid culture medium was removed, and the Matrigel dome was washed twice with DPBS. The Matrigel dome was treated with Cell Recovery Solution to completely remove the Matrigel, and total RNA was extracted using Trizol reagent (Ambion, CA). The RNA was converted to cDNA using reverse transcription reagents (TOYOBO, Japan) according to the manufacturer's protocol. The expression of mRNA was quantified using quantitative polymerase chain reaction with the SYBR Green Real-time PCR Master Mix Kit (TOYOBO). The reaction was performed on a Quant-Studio5 Real-Time PCR System (Applied Biosystem TM , CA) at the Soonchunhyang Biomedical Research Core Facility of the Korea Basic Science Institute. Target gene expression was calculated by comparing the relative expression levels after normalization to those of GAPDH. The primer sequences used are listed in Table 1.

Statistical analysis
Statistical significance between groups was assessed by the one-way of variance (ANOVA), using the GraphPad software (PRISM 8 Graphpad, CA). A p-value of ≤ 0.05 was considered statistically significant. All data presented in each experiment are representative results from three independent biological experiments. Data are presented as mean ± standard deviation, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.0005.

Vitamin D 3 induces small intestine IEC differentiation
Vitamin D 3 is known to induce cell differentiation of colorectal cancer-derived epithelial cell lines [7], and epidemiological studies showed that an increasing level of serum vitamin D 3 is positively correlated with colonic epithelial cell differentiation [13,28]. However, the direct effect of vitamin D 3 on small intestine IECs has not been studied. To determine whether vitamin D 3 influences the differentiation of IECs in the small intestine, we treated small intestinal organoids with various concentrations (10, 50, and 100 nM) of vitamin D 3, and the expression levels of specific IEC lineage markers were determined at 3 days post-treatment. The cell lineage markers comprised mucin (MUC2), lysozyme (LYZ), chromogranin A (CHGA), and villin (VIL), which are markers for goblet, Paneth, enteroendocrine cells and enterocytes, respectively. The expression level of MUC2 was significantly increased by vitamin D 3 at all treated concentrations (approximately 2-to 10-fold compared with that of the non-treated group). The expression levels of LYZ and VIL were significantly increased by 50 or 100 nM vitamin D 3 (approximately 2-to 4-fold compared with those of the non-treated group). Moreover, the expression level of CHGA was significantly upregulated by 100 nM vitamin D 3 (3-fold compared with that of the non-treated group) (Fig. 1a). To confirm the vitamin D 3 -induced increase in IEC differentiation, the numbers of specific differentiated IECs were examined using immunofluorescence staining. Consistent with the gene expression levels, the numbers of each differentiated IEC type increased with vitamin D 3 treatment, especially by treatment with 100 nM vitamin D 3 (Fig. 1b). The increased numbers of differentiated IECs were confirmed by the mean fluorescence intensities of all lineage markers, as presented in Figure S1A. The mean fluorescence intensities of Mucin 2 and Villin were significantly increased by treatment with 100 nM vitamin D 3 , while the intensities of lysozyme and chromogranin A were significantly elevated by treatment with both 50 and 100 nM vitamin D 3 . Taken together, these data suggest that vitamin D 3 induces general IEC differentiation in the small intestine.

Vitamin D 3 suppresses IEC stemness and proliferation
The effect of vitamin D 3 on IEC proliferation and stemness in the small intestine under normal conditions remains to be determined. To study the effect of vitamin D 3 on ISCs, small intestinal organoids were treated with various concentrations (10, 50, and 100 nM) of vitamin D 3, and the number of budding organoids was determined, as the crypt-villus formation of intestinal organoids originates from the renewal and proliferation of ISCs [27]. We found that the percentage of budding organoids was significantly reduced by 50 or 100 nM vitamin D 3 (Fig. 2a), suggesting the malfunction of ISCs upon vitamin D 3 treatment. To further determine whether the reduction in budding was caused by the depletion of ISCs or cell proliferation, the expression levels of LGR5 and Ki67 (markers for ISCs and cell proliferation, respectively) were quantified using qPCR. The expression levels of both LGR5 and Ki67 were dramatically downregulated by vitamin D 3 at all concentrations tested (Fig. 2b). Next, the intestinal organoids derived from LGR5-GFP-positive cells were utilized to confirm the depletion of ISCs and cell proliferation. After treatment with vitamin D 3 , the LGR5-GFP signal was observed under a fluorescence microscope, and Ki67-positive cells were evaluated using immunofluorescence staining. Consistent with the gene expression levels, the LGR5-GFP signal was negatively correlated with the vitamin D 3 concentration. Moreover, the number of Ki67-positive cells was also decreased by vitamin D 3 treatment, especially by 50 or 100 nM of vitamin D 3 (Fig. 2c and Figure  S1B), suggesting that vitamin D 3 suppresses IEC stemness and cell proliferation.
Notch signaling is known to regulate ISC function in the adult small intestine [29]. Furthermore, a previous study showed that the transcription activator brahma-  GCT GCC TGT AGT GTC AA  TCT TGA GGC TCG CCT TGA TG LGR5  related gene 1 (BRG1) plays a role in intestinal growth, crypt-villus formation, and stemness through the regulation of Notch1 signaling [30]. As our data showed that vitamin D 3 could suppress ISCs, we also quantified the expression of BRG1 and NOTCH1 in vitamin D 3 -treated organoids. We found that the expression levels of both BRG1 and NOTCH1 were significantly decreased by vitamin D 3 treatment (Fig. 2d). Taken together, these data suggest that vitamin D 3 causes ISC depletion and inhibits IEC proliferation in the small intestine.

Vitamin D 3 promotes apoptotic cell death in intestinal organoids
Previous studies showed that vitamin D 3 is associated with IEC survival [7,18]. However, there is no evidence showing the direct effect of vitamin D 3 on IECs in the small intestine, where vitamin D 3 is readily absorbed. As our results showed that vitamin D 3 could suppress IEC stemness and proliferation, we hypothesized that vitamin D 3 might alter intestinal viability due to the depletion of ISCs. Intestinal organoids were treated with various concentrations of vitamin D 3 , and organoid viability was assessed using the MTT assay. Viable cells reduced MTT to formazan, which is represented by the dark purple color in the organoids. The number of organoids containing formazan was decreased by vitamin D 3 treatment at all tested concentrations (Fig. 3a), suggesting that vitamin D 3 reduces IEC viability. As the association between apoptotic IEC death and VDR was previously described [18], we hypothesized that vitamin D 3-induced reduction in organoid viability might be mediated through apoptosis. Intestinal organoid apoptosis was determined using the TUNEL assay and immunofluorescence staining for cleaved caspase-3. The results showed The expression levels of leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) and Ki67, markers of ISCs and cell proliferation, respectively, were quantified using qPCR (b). Intestinal organoids derived from LGR5-green fluorescent protein (GFP) mice were treated with vitamin D 3. The number of LGR5-GFP-positive cells was visualized using a confocal microscope (green fluorescence), and the number of Ki67positive cells was determined following immunofluorescence staining (red fluorescence) (c). The expression levels of Brahma-related gene 1 (BRG1) and NOTCH 1 were quantified using qPCR (d). Data are presented as mean ± standard deviation, ***p ≤ 0.0005 that the signal was dramatically increased following 50 or 100 nM vitamin D 3 treatment in intestinal organoids treated with the TUNEL reagents (Fig. 3b). In addition, the immunofluorescence signal of cleaved caspase-3 was also increased following 50 or 100 nM vitamin D 3 treatment ( Fig. 3c and Figure S1C). Taken together, these data confirm that high levels of vitamin D 3 promote apoptotic cell death in the small intestine.

Vitamin D 3 suppresses stemness by augmenting ER stress
ER stress augmentation can lead to ISC loss and induce IEC differentiation by UPR activation [25]. Furthermore, UPR is involved in cell survival [23,24]. As stemness and viability were reduced in vitamin D 3 -treated organoids, we hypothesized that the action of vitamin D 3 on stemness and viability might be mediated by ER stress induction. First, we determined whether ER stress is induced by vitamin D 3 treatment. The expression levels of UPR genes, the C/EBP homologous protein (CHOP), the activating transcription factor 6 (ATF6), and the X-boxbinding protein 1 (XBP1) in either total form (tXBP1) or spliced form (sXBP1) were quantified using qPCR. The results showed that the expression level of CHOP was significantly increased by vitamin D 3 at all tested concentrations. The expression level of ATF6 was significantly upregulated by vitamin D 3 at 50 or 100 nM. In addition, the expression level of tXBP1 was significantly increased by 100 nM vitamin D 3 ; however, the expression of sXBP1 did not change with vitamin D 3 treatment (Fig. 4a), suggesting that D 3 treatment induces ER stress. Secondly, we determined whether ER stress induction mediates the action of vitamin D 3 on IEC stemness and apoptotic cell death. According to our results, 50 nM vitamin D 3 is the minimum concentration that induces UPR gene expression, reduces stemness, and promotes apoptotic cell death. Therefore, intestinal organoids were  (10,50, and 100 nM) of vitamin D 3 . The percentage of viability was evaluated by the reduction of the MTT reagent (a). Apoptotic cells were identified using the TUNEL assay (red fluorescence) (b) and immunofluorescence staining for cleaved caspase-3 (green fluorescence) (c). Data are presented as mean ± standard deviation, **p ≤ 0.01, ***p ≤ 0.0005 treated with 50 nM vitamin D 3 in the presence or absence of 250 μM or 500 μM TUDCA, a classical ER stress inhibitor, and budding was observed. The results showed that the percentage of budding was significantly rescued in the presence of 500 μM TUDCA (Fig. 4b), which might be a result of stemness induction. To confirm that ER stress inhibition could rescue IEC stemness, the expression level of LGR5 was quantified using qPCR, and the signal from GFP was visualized using a confocal microscope. We found that the expression level of LGR5 was significantly upregulated in the presence of TUDCA (Fig. 4c). In addition, the mean fluorescence intensity of LGR5-GFP was partially rescued by TUDCA treatment (Fig. 4d and Figure S2A). Lastly, immunofluorescence staining of cleaved caspase-3 showed that vitamin D 3 treatment in the presence of TUDCA could reduce the cleaved caspase-3 signal compared with that of vitamin D 3 treatment alone (Fig. 4e and Figure S4B), suggesting Fig. 4 The effect of vitamin D 3 on small intestinal organoids is mediated by endoplasmic reticulum (ER) stress induction. Intestinal organoids were treated with various concentrations (10, 50, and 100 nM) of vitamin D 3 and the expression levels of unfolded protein response (UPR) genes, C/EBP homologous protein (CHOP), activating transcription factor 6 (ATF6), total form X-box-binding protein 1 (tXBP1), spliced form (s)XBP1 were quantified using qPCR (a). Small intestinal organoids derived from leucine-rich repeat-containing G-protein-coupled receptor 5-green fluorescent protein (LGR5-GFP) mice were treated with 50 nM vitamin D 3 in the presence or absence of the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) (250 or 500 nM). The budding of intestinal organoids was observed under a microscope (left panel), and the percentage of budding organoids was analyzed using the ImageJ software (right panel) (b). The expression level of LGR5 was quantified using qPCR (c), and the numbers of LGR5-GFP-positive cells were visualized using a confocal microscope (green fluorescence) (d). Immunofluorescence images of cleaved caspase-3 (green fluorescence) (e). Data are presented as mean ± standard deviation, *p ≤ 0.05, ***p ≤ 0.0005 that the effect of vitamin D 3 on apoptotic cell death is mediated by ER stress induction. Furthermore, we did not observe changes in Ki67, LYZ, and MUC2 expression levels, while the expression levels of CHGA and VIL were significantly increased in the presence of 250 or 500 μM, and 500 μM TUDCA, respectively ( Figure  S2C). Thus, our findings suggest that vitamin D 3 suppresses IEC stemness and promotes apoptotic cell death partially through ER stress activation.

Discussion
Vitamin D 3 plays an important role in many biological processes, such as intestinal calcium absorption, maintenance of intestinal epithelial integrity and function, and cancer suppression [3,7,8]. However, the levels of vitamin D 3 in the body should be well regulated to avoid side effects. For example, vitamin D 3 deficiency could lead to disease development, such as inflammatory bowel disease and pancreatitis [9]. In contrast, excess vitamin D 3 levels could alter the intestinal microbiota composition and increase disease susceptibility [10]. Most studies of vitamin D 3 effects on IECs have been conducted using colorectal cancer cell lines and in vivo mouse models, specifically under disease induction, such as colitis, mainly focusing on the colon [7,13,20]. As vitamin D 3 is mainly absorbed in the small intestine, the effect of vitamin D 3 on IECs in the small intestine should be understood.
Many IEC cell lineages are generated from ISCs, including secretory cells, such as Paneth (lysozyme-producing cells), goblet (mucin-producing cells), and enteroendocrine cells, as well as absorptive enterocytes [12]. ISCs undergo proliferation and differentiation for homeostatic turnover of the intestinal epithelium and ensure epithelial regeneration following intestinal damage [31,32]. Previous studies showed that vitamin D 3 influences the proliferation and differentiation [7,33] as well as the survival of IECs in the colon [34]. Therefore, we hypothesized that vitamin D 3 might also affect the differentiation, proliferation, stemness, and survival of IECs in the small intestine under normal conditions. We used intestinal organoids to assess the effect of vitamin D 3 on IECs in the small intestine. Consistent with the effects of vitamin D 3 on colonic IECs [7,18], we demonstrated that vitamin D 3 globally induced IEC differentiation into specific cell lineages, including goblet, Paneth, enteroendocrine cells, and enterocytes, represented by the increased expression of mucin-2, lysozyme, chromogranin A, and villin, respectively. It is well known that cell differentiation and proliferation are coordinately regulated by the growth factors present in the microenvironment, including those in the small intestine [35]. While vitamin D 3 increased cell differentiation, it drastically suppressed IEC proliferation, represented by the reduction in Ki67-positive cells in intestinal organoids.
As ISCs are highly proliferative cells [36], we hypothesized that the decrease in IEC proliferation might be due to the suppression of ISCs. Unlike normal colon organoids derived from humans, where vitamin D 3 upregulates stem cell-related genes [33], our data showed that vitamin D 3 inhibited the expression level of LGR5 and decreased the number of LGR5-GFP-positive cells in small intestinal organoids derived from LGR5-GFP mice, suggesting that vitamin D 3 reduces stemness in the small intestine. Moreover, we revealed the downregulation of the gene expression levels of BRG1 and Notch1, which are also known to regulate ISC function and IEC differentiation [29,30], as well as the reduction of budding organoids, which support the effect of vitamin D 3 on ISC depletion. However, direct evidence showing that vitamin D 3 either influences stemness maintenance or proliferation needs to be demonstrated in further studies. The discrepancy between the effects of vitamin D 3 on the organoids derived from the small intestine and colon might be explained by several possibilities, including the components in the crypt base compartment, the proliferative rate as well as the distinct molecular signature, and intrinsic regulation in stem cell population [37,38]. Notably, vitamin D 3 concentrations might be important for the effect of vitamin D 3 on IECs in the small intestine. Previous studies showed that vitamin D 3 can reduce the viability of colorectal carcinoma cell lines via the induction of apoptosis in a dose-dependent manner [16,17]; therefore, we hypothesized that the depletion of intestinal stemness by vitamin D 3 may be associated with apoptosis-induced cell death. Our results showed that vitamin D 3 promotes apoptotic cell death, especially at high doses. In addition, our study may support a potential biological significance of vitamin D 3 in cancer stem cell therapy, which has been reviewed previously [39,40]. Vitamin D 3 may have a probable beneficial role in the inhibition of progression and survival, as well as in facilitating the apoptosis of cancer stem cells, resulting in reduction of the self-renewal capacity that initiates tumor formation. However, whether apoptotic cell death mainly occurs in ISCs, proliferative cells, or differentiated cells remains to be determined. Therefore, the specific IEC lineage targeted by vitamin D 3 needs to be identified.
ER stress induction is known to be related with cell survival or apoptotic cell death, depending on the severity of ER stress [23,24]. Furthermore, the activation of UPR upon ER stress induction is associated with the functions of goblet and Paneth cells and is important for the differentiation of ISCs into TA cells [25,26]. Therefore, we hypothesized that the effect of vitamin D 3 on IEC stemness and survival in the small intestine might be mediated by ER stress induction. Our results showed that the expression levels of UPR genes, especially those of C/EBP homologous protein (CHOP) and activating transcription factor 6 (ATF6), were upregulated in vitamin D 3 -treated organoids. However, treatment with TUDCA, which is known to reduce experimental colitis by abolishing ER stress in colonocytes [41], partially rescued the vitamin D 3 -induced depletion of LGR5-GFPpositive cells and reduced the number of cleaved caspase-3-positive cells in intestinal organoids, suggesting that the depletion of stemness and apoptotic cell death induction by vitamin D 3 may be mediated by ER stress induction. Although our findings showed that ER stress is partially involved in the depletion of stemness and induction of apoptotic cell death, additional mechanisms are likely involved, which remain to be identified. Our study provides evidence that vitamin D 3 alters the proliferation, differentiation, stemness, and survival of IECs in the small intestine. Moreover, the effect of vitamin D 3 on IEC stemness and survival is partially mediated by ER stress induction.

Conclusions
In summary, this study revealed that vitamin D 3 could induce cell differentiation, promote apoptotic cell death, and suppress cell proliferation and stemness in the small intestine partially through the activation of ER stress. These effects are similar to those of drugs that regulate cell survival via the activation of ER stress [42]. Future studies are required to determine the detailed mechanism underlying the regulation of IEC function by vitamin D 3 , specifically that of ISCs, which is important for controlling the levels of vitamin D 3 and maintaining intestinal homeostasis.