Imbalance of autophagy and apoptosis in intestinal epithelium lacking the vitamin D receptor

Abstract

Apoptosis and autophagy are dynamic processes that determine the fate of cells. Vitamin D receptor (VDR) deficiency in the intestine leads to abnormal Paneth cells and impaired autophagy function. Here, we will elucidate the mechanisms of the intestinal epithelial VDR regulation of autophagy and apoptosis. We used in vivo VDR^lox and VDR^∆IEC mice and ex vivo organoids generated from small intestine and colon tissues. We found that VDR deficiency induced more apoptotic cells and significantly increased cell death in the small intestine and colon of VDR^∆IEC mice. The proapoptotic protein B-cell lymphoma 2 (BCL-2) associated X protein (Bax) was enhanced, whereas autophagy related 16 like 1 (ATG16L1) and Beclin-1 were decreased in the intestines of VDR^ΔIEC mice. Apoptosis induced by Bax reduced autophagy by decreasing Beclin-1. Physical interactions between Beclin-1 and Bcl-2 were increased in the VDR-deficient epithelia from mice. The growth of VDR^∆IEC organoids was significantly slower with fewer Paneth cells than that of VDR^+/+ organoids. The expression levels of Beclin-1 and lysozyme were decreased in VDR^∆IEC organoids. Bacterial endotoxin levels were high in the serum from VDR^∆IEC mice and made mice susceptible to colitis. In the organoids and colitis IL-10^-/- mice, vitamin D₃ treatment increased VDR and ATG16L1 protein expression levels, which activated autophagic responses. In summary, intestinal epithelial VDR regulates autophagy and apoptosis through ATG16L1 and Beclin-1. Our studies provide fundamental insights into the tissue-specific function of VDR in modulating the balance between autophagy and apoptosis.-Lu, R., Zhang, Y.-G., Xia, Y., Sun, J. Imbalance of autophagy and apoptosis in intestinal epithelium lacking the vitamin D receptor.

Keywords: Beclin-1; VDR; colonoids; enteroids; inflammation.

Figure 1

VDR deficiency increased the number of apoptotic cells in VDR^∆IEC mice. A) Apoptotic cells were determined with TUNEL assays. Small intestines from VDR^lox and VDR^∆IEC mice: TUNEL signals (green) are indicated by the yellow arrows. Colon tissues from the VDR^lox and VDR^∆IEC mice: TUNEL signaling (green) was counterstained with DAPI nuclear stain (blue). B) A significant increase in the number of apoptotic cells in the villi was noted in VDR^∆IEC mice compared with that in the VDR^lox mice. The data are expressed as means ± sem (n = 6; Student’s t test). *P < 0.05, **P < 0.01. C) ELISA was used to quantitate cell death, and we found significantly increased cell death in the intestines of VDR^∆IEC mice. Intestinal tissues were harvested from VDR^lox and VDR^∆IEC mice; then, cell death was determined with ELISA kits according to the manufacturer’s instructions. The data are expressed as means ± sem; Student’s t test (small intestine, n = 9; colon, n = 5). *P < 0.05.

Figure 2

Apoptosis- and autophagy-related genes were regulated by intestinal epithelial VDR. A) Enhanced cleaved caspase-3 in VDR^∆IEC mice according to immunohistochemistry staining. Representative images of cleaved caspase-3 immunohistochemistry in small intestinal crypts from VDR^lox and VDR^∆IEC mice. B) Western blots for Beclin-1, Bcl-2, Bcl-xL, Bax, P62, LC3I/II, PUMA, and cleaved caspase-3 in the small intestines of VDR^∆IEC and VDR^lox mice. We found reduced levels of Beclin-1 and enhanced levels of Bcl-2, Bcl-xL, P62, and cleaved caspase-3. C) The intensity of gene expression in the intestinal tissues was analyzed by Western blotting, and the ratio of their expression to that of β-actin was calculated. D) Real-time PCR for Beclin-1, Bcl-2, Bcl-xL, Bax, LYZ, and ATG16L1 in the intestines of VDR^∆IEC and VDR^lox mice. LC3, autophagy-related proteins, light chain 3; LYZ, lysozyme. The data represent means ± sem; Student’s t test (n  = 5 mice/group). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Intestinal VDR affected the Bcl-2 and Beclin-1 complex interaction. A) Bcl-2 and Beclin-1 interaction in VDR^lox and VDR^∆IEC mice was detected by immunoprecipitation. Physical interactions between Beclin-1 and Bcl-2 were increased in the VDR-deficient epithelia from mice. B) The gene expression levels of Bcl-2 and Beclin-1 in the enteroids from VDR^lox and VDR^∆IEC enteroids were detected by costaining. We identified increased Bcl-2 and Beclin-1 colocalization in the VDR^ΔIEC mice according to immunofluorescence staining. IP, immunoprecipitation; Wb, Western blot.

Figure 4

Growth was significantly slower in VDR^∆IEC organoids than in VDR^lox organoids. A) The growth of VDR^∆IEC and VDR^lox organoids from the small intestine and colon. B) The number of organoids formed from 1 × 10⁵ single crypt cells was used to calculate the efficiency. The organoid-forming efficiency of the VDR^∆IEC group relative to the VDR^lox control group after 10 d is shown. The data represent the mean ± sem of 3 independent experiments. Statistical analysis was performed by 1-way ANOVA (n  =  6/group). *P < 0.05, **P < 0.01. C) Protein expression in VDR^∆IEC and VDR^lox organoids according to Western blotting. Decreased lysozyme, Beclin-1, and ATG16L1 protein levels were found in the VDR^∆IEC mice compared with the VDR^lox group. D) The intensity of gene expression was analyzed by Western blotting, and the ratio of their expression to that of β-actin was calculated. The data represent means ± sem; Student’s t test (n  = 5/group). *P < 0.05, **P < 0.01. E) BrdU staining in the enteroids. Proliferation was insignificantly lower in the VDR^∆IEC enteroids. D, day. The data represent means ± sem; Student’s t test (n = 10/group). ***P < 0.001.

Figure 5

Specialized epithelial cells in VDR^∆IEC enteroids. A) Detection of the goblet cell marker Mucin2 (green) in enteroids. B) Enteroids were treated with 50 μm Z-VAD-FMK (apoptosis inhibitor) for 72 h, and then the expression of Mucin2 (green) were detected. C) Detection of the autophagy downstream marker lysozyme (red) in enteroids. D) Lysozyme and Mucin2 expression were lower in VDR^∆IEC organoids than in VDR^lox organoids. The data represent means ± sem; Student’s t test (n = 10/group). *P < 0.05, **P < 0.01. E) More bacterial endotoxin was found in serum samples from VDR^∆IEC mice. Serum samples from VDR^lox and VDR^∆IEC mice were measured for bacterial endotoxin levels with LAL chromogenic endpoint assays. The data are expressed as means ± sem. (n = 8; Student’s t test). *P < 0.05. F) The expression level of Lcn-2 was higher in VDR^∆IEC mice than in VDR^lox mice. Student’s t test (n = 10/group). *P < 0.05. G) Vitamin D enhanced autophagy and reduced apoptosis in VDR^+/+ organoids. Enteroids from VDR^+/+ mice were treated with 20 nM vitamin D₃ for 3 and 24 h. Increased VDR and ATG16L1 protein levels were found in VDR^+/+ organoids treated with vitamin D. H, I) The intensity of gene expression in the intestinal tissues was analyzed by Western blotting, and the ratio of their expression to that of LC3I/II (H) or β-actin (I) was calculated. The data represent means ± sem; Student’s t test (n = 3/group). *P < 0.05, **P < 0.01. J) Enteroids from VDR^+/+ mice were treated with 20 nM vitamin D₃ for 3 h and then added with 0.5 ng/ml TNFα for 30 min. Vitamin D₃ (VitD3) can increase the expression of VDR and affect the expression of IκBα. K) TUNEL assay of VDR^∆IEC enteroids treated with 100 nM rapamycin for 24 h. Apoptotic cells per enteroids were accounted. LC3, autophagy-related proteins, light chain 3. Data are expressed as means ± sem (n = 30 enteroids).

Figure 6

VDR in DSS-induced colitis models. A) VDR^lox and VDR^ΔIEC mice were administrated with 5% DSS. Representative hematoxylin and eosin histology of colon from VDR^lox and VDR^ΔIEC mice with or without DSS treatment. B) Inflammatory scores of the mouse intestine showed that VDR^ΔIEC mice were susceptible to chemical injury (n = 5/group). *P < 0.05 compared with VDR^lox mice. C) Western blots for Beclin-1, Bcl-2, ATG16L1, and cleaved caspase-3 in the intestines of VDR^∆IEC and VDR^lox mice. D) IL-10^−/− mice were gavaged by 0.2 μg 1,25D₃ in 0.1 ml corn oil 3 times per week for 4 wk. Colon tissues were collected for Western blotting. Vitamin D treatment could increase the expression levels of VDR, ATG16L1, and Beclin-1 in the IL-10^−/− mice.

Figure 7

A working model of intestinal epithelial VDR in apoptosis and autophagy balance. The VDR controls the autophagy and apoptosis checkpoint in IECs by maintaining the expressions of Beclin-1 and ATG16L1. VDR deficiency leads to impaired autophagy and enhanced cell death, thus increasing serum LPS and promoting inflammation.