Epithelial Vitamin D Receptor Research Articles

Intestinal vitamin D receptor protects against extraintestinal breast cancer tumorigenesis

ABSTRACT The microbiota plays critical roles in regulating the function and health of the intestine and extraintestinal organs. A fundamental question is whether an intestinal-microbiome-breast axis exists during the development of breast cancer. If so, what are the roles of host factors? Vitamin D receptor (VDR) involves host factors and the human microbiome. Vdr gene variation shapes the human microbiome, and VDR deficiency leads to dysbiosis. We hypothesized that intestinal VDR protects hosts against tumorigenesis in the breast. We examined a 7,12-dimethylbenzanthracene (DMBA)-induced breast cancer model in intestinal epithelial VDR knockout (VDRΔIEC) mice with dysbiosis. We reported that VDRΔIEC mice with dysbiosis are more susceptible to breast cancer induced by DMBA. Intestinal and breast microbiota analysis showed that VDR deficiency leads to a bacterial profile shift from normal to susceptible to carcinogenesis. We found enhanced bacterial staining within breast tumors. At the molecular and cellular levels, we identified the mechanisms by which intestinal epithelial VDR deficiency led to increased gut permeability, disrupted tight junctions, microbial translocation, and enhanced inflammation, thus increasing tumor size and number in the breast. Furthermore, treatment with the beneficial bacterial metabolite butyrate or the probiotic Lactobacillus plantarum reduced breast tumors, enhanced tight junctions, inhibited inflammation, increased butyryl-CoA transferase, and decreased levels of breast Streptococcus bacteria in VDRΔIEC mice. The gut microbiome contributes to the pathogenesis of diseases not only in the intestine but also in the breast. Our study provides insights into the mechanism by which intestinal VDR dysfunction and gut dysbiosis lead to a high risk of extraintestinal tumorigenesis. Gut-tumor-microbiome interactions represent a new target in the prevention and treatment of breast cancer.

Vitamin D-VDR (vitamin D receptor) alleviates glucose metabolism reprogramming in lipopolysaccharide-induced acute kidney injury.

Background: Our previous study showed that vitamin D (VD)-vitamin D receptor (VDR) plays a nephroprotective role in lipopolysaccharide (LPS)-induced acute kidney injury (AKI). Recently, glucose metabolism reprogramming was reported to be involved in the pathogenesis of AKI. Objective: To investigate the role of VD-VDR in glucose metabolism reprogramming in LPS-induced AKI. Methods: We established a model of LPS-induced AKI in VDR knockout (VDR-KO) mice, renal proximal tubular-specific VDR-overexpressing (VDR-OE) mice and wild-type C57BL/6 mice. In vitro, human proximal tubular epithelial cells (HK-2 cells), VDR knockout and VDR overexpression HK-2 cell lines were used. Results: Paricalcitol (an active vitamin D analog) or VDR-OE reduced lactate concentration, hexokinase activity and PDHA1 phosphorylation (a key step in inhibiting aerobic oxidation) and simultaneously ameliorated renal inflammation, apoptosis and kidney injury in LPS-induced AKI mice, which were more severe in VDR-KO mice. In in vitro experiments, glucose metabolism reprogramming, inflammation and apoptosis induced by LPS were alleviated by treatment with paricalcitol or dichloroacetate (DCA, an inhibitor of p-PDHA1). Moreover, paricalcitol activated the phosphorylation of AMP-activated protein kinase (AMPK), and an AMPK inhibitor partially abolished the protective effect of paricalcitol in LPS-treated HK-2 cells. Conclusion: VD-VDR alleviated LPS-induced metabolic reprogramming in the kidneys of AKI mice, which may be attributed to the inactivation of PDHA1 phosphorylation via the AMPK pathway.

Vitamin D receptor upregulates tight junction protein claudin-5 against colitis-associated tumorigenesis.

Tight junctions are essential for barrier integrity, inflammation, and cancer. Vitamin D and the vitamin D receptor (VDR) play important roles in colorectal cancer (CRC). Using the human CRC database, we found colonic VDR expression was low and significantly correlated with a reduction of Claudin-5 mRNA and protein. In the colon of VDRΔIEC mice, deletion of intestinal VDR led to lower protein and mRNA levels of Claudin-5. Intestinal permeability was increased in the VDR−/− colon cancer model. Lacking VDR and a reduction of Claudin-5 are associated with an increased number of tumors in the VDR−/− and VDRΔIEC mice. Furthermore, gain and loss functional studies have identified CLDN-5 as a downstream target of VDR. We identified the Vitamin D response element (VDRE) binding sites in a reporter system showed that VDRE in the Claudin-5 promoter is required for vitamin D3-induced Claudin-5 expression. Conditional epithelial VDR overexpression protected against the loss of Claudin-5 in response to inflammation and tumorigenesis in vivo. We also reported fecal VDR reduction in a colon cancer model. This study advances the understanding of how VDR regulates intestinal barrier functions in tumorigenesis and the possibility for identifying new biomarker and therapeutic targets to restore VDR-dependent functions in CRC.

Vitamin D receptor promotes healthy microbial metabolites and microbiome

Microbiota derived metabolites act as chemical messengers that elicit a profound impact on host physiology. Vitamin D receptor (VDR) is a key genetic factor for shaping the host microbiome. However, it remains unclear how microbial metabolites are altered in the absence of VDR. We investigated metabolites from mice with tissue-specific deletion of VDR in intestinal epithelial cells or myeloid cells. Conditional VDR deletion severely changed metabolites specifically produced from carbohydrate, protein, lipid, and bile acid metabolism. Eighty-four out of 765 biochemicals were significantly altered due to the Vdr status, and 530 significant changes were due to the high-fat diet intervention. The impact of diet was more prominent due to loss of VDR as indicated by the differences in metabolites generated from energy expenditure, tri-carboxylic acid cycle, tocopherol, polyamine metabolism, and bile acids. The effect of HFD was more pronounced in female mice after VDR deletion. Interestingly, the expression levels of farnesoid X receptor in liver and intestine were significantly increased after intestinal epithelial VDR deletion and were further increased by the high-fat diet. Our study highlights the gender differences, tissue specificity, and potential gut-liver-microbiome axis mediated by VDR that might trigger downstream metabolic disorders.

Myeloid vitamin D receptor influences cell proliferation in small intestine

The vitamin D receptor (VDR) is a ubiquitous nuclear receptor that acts as a transcription factor when vitamin D binds with VDR. Intestinal epithelial VDR has various physiological and anatomical benefits to intestine. Interactions between intestinal epithelial cells (IEC) and immune cells are critical for gut health. Cellular proliferation through Wnt/β‐catenin signaling is a result of immune and IEC crosstalk. Wnt proteins from macrophages bind to intestinal epithelial cells and activate the Wnt/β‐catenin pathway. This pathway is critical for cell proliferation which enables food and water absorption in the small intestine. However, the importance of myeloid VDR in regulating the Wnt/β‐catenin pathway in epithelial cells is not fully understood. The objective of this study is to assess how the presence of myeloid VDR in the intestinal epithelial cells influences cellular proliferation through the Wnt/β‐catenin pathway. We hypothesize that the low level of myeloid VDR will reduce expression of Wnt/β‐catenin signaling downstream and alleviate cell proliferation in the small intestine. This experiment utilized selective deletion of myeloid VDR in an ex‐vivo and in‐vivo set‐up. For the ex‐vivo, control mice (VDRLoxP) were crossed with lysozyme (M) ‐cre mice to create specific knockout of myeloid VDR (VDRrLyz) mice. Twelve mice (6 VDRLoxP mice and 6 VDRrLyz) were sacrificed and mucosal scrapings from the ileum (small intestine) were collected for westerns. For cell culture, small intestine organoids derived from the intestinal stem cells were cultured alone or co‐cultured with macrophages (VDR+/+ or VDR−/−). After 3 days, the organoids and organoid‐macrophage co‐cultures were collected for western blots. Western blots were used to measure and compare expression of Wnt/β‐catenin signaling proteins. The VDR−/− organoid macrophage co‐culture showed significantly decreased expression of β‐catenin and PCNA (a cell proliferation marker), compared to the VDR+/+ organoid macrophage co‐culture. Our results suggest that myeloid VDR influences the Wnt/β‐catenin signaling pathway. We present a novel role of myeloid VDR signaling in mediating immune/epithelial cell cross talk in intestine.

Lactic Acid Bacteria Isolated From Korean Kimchi Activate the Vitamin D Receptor–autophagy Signaling Pathways

Probiotic lactic acid bacteria (LAB) have been used in the anti-inflammation and anti-infection process of various diseases, including inflammatory bowel disease (IBD). Vitamin D receptor (VDR) plays an essential role in pathogenesis of IBD and infectious diseases. Previous studies have demonstrated that the human VDR gene is a key host factor to shape gut microbiome. Furthermore, intestinal epithelial VDR conditional knockout (VDRΔIEC) leads to dysbiosis. Low expressions of VDR is associated with impaired autophagy, accompanied by a reduction of ATG16L1 and LC3B. The purpose of this study is to investigate probiotic effects and mechanism in modulating the VDR-autophagy pathways. Five LAB strains were isolated from Korean kimchi. Conditional medium (CM) from these strains was used to treat a human cell line HCT116 or intestinal organoids to measure the expression of VDR and autophagy. Mouse embryonic fibroblast (MEF) cells with or without VDR were used to investigate the dependence on the VDR signaling. To test the role of LAB in anti-inflammation, VDR+/+ organoids were treated with 121-CM before infection with Salmonella enterica serovar Enteritidis. In vivo, the role of LAB in regulating VDR-autophagy signaling was examined using LAB 121-CM orally administrated to VDRLoxp and VDRΔIEC mice. The LAB-CM-treated groups showed higher mRNA expression of VDR and its target genes cathelicidin compared with the control group. LAB treatment also enhanced expressions of Beclin-1 and ATG16L1 and changed the ratio of LC3B I and II, indicating the activation of autophagic responses. Furthermore, 121-CM treatment before Salmonella enterica serovar Enteritidis infection dramatically increased VDR and ATG16L1 and inhibited the inflammation. Administration of 121-CM to VDRLoxp and VDRΔIEC mice for 12 and 24 hours resulted in an increase of VDR and LC3B II:I ratio. Furthermore, we identified that probiotic proteins P40 and P75 in the LAB-CM contributed to the anti-inflammatory function by increasing VDR. Probiotic LAB exert anti-inflammation activity and induces autophagy. These effects depend on the VDR expression. Our data highlight the beneficial effects of these 5 LAB strains isolated from food in anti-infection and anti-inflammation.

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

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 VDRlox 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 VDRΔIEC 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.

Targeting Intestinal Vitamin D Receptor Signaling to Mitigate Graft-Versus-Host Disease

Graft-versus-host disease (GVHD) remains the major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Impaired mucosal epithelial barrier contributes significantly to amplifying systemic inflammatory responses characteristic of GVHD. Emerging evidence demonstrates that protecting or restoring intestinal barrier function represents a feasible approach to mitigate GVHD.There is growing interest in how vitamin D and the vitamin D receptor (VDR) signaling regulate immune responses in intestinal inflammatory diseases. The role of VDR signaling in regulating alloimmunity is unknown. We therefore tested the novel hypothesis that VDR signaling plays an important role in maintaining intestinal epithelial barrier function during GVHD. In initial studies, we examined whether intestinal VDR signaling is altered in recipient mice undergoing either syngeneic or allogeneic bone marrow transplantation (BMT). We found a progressive decline in VDR gene expression over time in colon tissues of allogeneic, but not syngeneic, BMT recipients compared with that of naïve animals. Notably, the expression of CYP27B1, the key enzyme participating in the biosynthesis of the active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25VitD3), was also significantly decreased in the intestines of allogeneic BMT recipients compared to that of naïve animals. Caco-2 is an epithelial cell line that is widely used as a model for the intestinal barrier. We showed that proinflammatory cytokines TNF-α and IL-6 reduced Caco-2 VDR protein levels in a dose and time dependent manner by western blot analysis. To assess monolayer barrier function, an in vitro permeability assay was performed using FITC-dextran. We found 1,25VitD3 significantly attenuated the increase in epithelial monolayer permeability induced by TNF-α and IL-6. Tight junctions are important components of the intestinal barrier. We also found proinflammatory cytokines reduced gene expression of tight junctions such as occludin in Caco-2 cells. Remarkably, 1,25VitD3 treatment significantly restored occludin expression.We then examined how vitamin D/VDR pathway regulates the development of GVHD. 1,25VitD3 supplementation after BMT significantly increased the survival of recipient mice in a well-established C57Bl/6→Balb/c GVHD model (p=0.0045). 1,25VitD3 treatment did not appear to modify donor T cell alloreactivity. Specifically, donor T cell activation, proliferation, and polarization were not affected by 1,25VitD3 treatment. Notably, infusion of donor T cells from VDR-WT (VDR+/+) or VDR-deficient (VDR-/-) mice caused similar GVHD-associated mortality in Balb/c recipient mice (p=0.98), further suggesting that the beneficial effects of 1,25VitD3 treatment is not due to its effects on donor T cells. 1,25VitD3 also did not appear to affect host and donor antigen presenting cells to modulate GVHD severity. Specifically, dendritic cells from VDR+/+ or VDR-/- mice showed equal ability to stimulate the proliferation of alloreactive T cells in mixed lymphocyte reactions. There was also no difference in post-transplant survival when recipient mice were reconstituted with donor T-cell-depleted bone marrow cells from either VDR+/+ or VDR-/- mice (recipients were transplanted with the same donor T cells from VDR-WT animals; p=0.58). Instead, 1,25VitD3 treatment significantly increased tight junction protein expression in the gut, suggesting its effect on intestinal epithelial cells. Finally, transgenic mice selectively overexpress VDR in the intestinal epithelium (villin promoter-driven FLAG-tagged human VDR gene expression) were used as BMT recipients in gain-of-function studies. We found a significantly decreased GVHD-associated mortality in these mice compared to that of control littermates (p=0.0002), providing direct evidence that intestinal epithelial VDR signaling protects against GVHD. Our results provide new evidence supporting the notion that impaired mucosal barrier function is a major pathogenic factor in the development of GVHD. Intestinal VDR signaling plays an important role in maintaining intestinal barrier integrity and enhancing this pathway protects against GVHD. Thus, our study provides pre-clinical data supporting the use of vitamin D or its analogs as a simple and inexpensive therapeutic modality with minimal side effects for mitigating GVHD. DisclosuresNo relevant conflicts of interest to declare.

Lack of Vitamin D Receptor Leads to Hyperfunction of Claudin-2 in Intestinal Inflammatory Responses.

Vitamin D3 and vitamin D receptor (VDR) are involved in the pathogenesis of inflammatory bowel disease (IBD) and bacterial infection. Claudin-2 is a junction protein that mediates paracellular water transport in epithelia. Elevation of Claudin-2 is associated with active IBD. However, VDR involved in epithelial junctions under inflammation and infection remains largely unknown. We investigated the mechanisms on how VDR and Claudin-2 are related in inflamed states. Using cultured VDR-/- enteroids, human intestinal epithelial cells, VDR-/- mice with Salmonella- or DSS-colitis, and human IBD samples, we investigated the mechanisms how VDR and Claudin-2 are related in inflamed states. After Salmonella infection had taken place, we observed significantly enhanced Claudin-2 and an increased bacterial invasion and translocation. A lack of VDR regulation led to a robust increase of Claudin-2 at the mRNA and protein levels post-infection. In DSS-treated VDR-/- mice, Claudin-2 was significantly increased. Location and quantification of Claudin-2 protein in the mouse colons treated with DSS also confirmed these results. Inflammatory cytokines were significantly higher in the serum and mRNA levels in intestine, which are known to increase Claudin-2. Furthermore, in inflamed intestine of ulcerative colitis patients, VDR expression was low and Claudin-2 was enhanced. Mechanistically, we identified the enhanced Claudin-2 promoter activity through the binding sites of NF-κB and STAT in inflamed VDR-/- cells. Our studies have identified a new role for intestinal epithelial VDR in regulating barrier functions in the context of infection and inflammation.

Abstract 5215: Vitamin D receptor regulates intestinal barriers in colon cancer

Abstract Background Vitamin D and Vitamin D receptor (VDR) play important roles in the development of colon cancer. Tight junction structures are essential in intestinal barrier integrity, inflammation, and cancer. The disruption of tight junctions is a common manifestation of various diseases including cancers. Claudin-5 is a tight junction protein that is expressed in epithelia and endothelia and form paracellular barriers and pores that determine permeability. It is downregulated in colon cancer. Although VDR and Claudin-5 are all involved in colon cancer, it remains unclear whether they are closely related or function independently. Methods In the current study, we investigate the novel role of VDR in regulating Claudin-5, using whole body VDR knockout mice (VDR-/-) and intestinal epithelial VDR knockout mice (VDRΔIEC). In vitro, VDR-/- embryonic fibroblasts (MEF) cells, cultured human intestinal epithelial cells, organoids, and human colon cancer samples were used to determine the molecular mechanism. Results In intestinal samples of colon cancer patients, VDR expression is low, whereas the expression of Claudin-5 is also decreased. In the colon of VDR-/- mice and VDRΔIEC mice, deletion of VDR led to lower claudin-5 at the protein and mRNA levels. This observation was also confirmed by immunostaining of Claudin-5. In VDR-/- MEF cells, both Western blot and real time PCR revealed that VDR deletion led to lower protein and mRNA levels of claudin-5 in cells without any treatment. Vitamin D3 is known to increase VDR expression and activate VDR signaling. Protein and mRNA levels of Claudin-5 were significantly elevated in mice, mouse organoids, and human intestinal epithelial SKCO15 cells treated with 1, 25 vitamin D3. In contrast, knockdown of Claudin-5 using siRNA did not change the expression level of VDR protein in human colonic epithelial cells. Our data suggest that the Claudin-5 gene is a downstream target of the VDR signaling pathway. Conclusion Taken together, we show that VDR is important for the maintenance of physiological level of Claudin-5. This study also reveals a complex role for vitamin D/VDR regulation of intestinal barrier functions in colon cancer. Citation Format: Yong-guo Zhang, Rong Lu, Danika Bakke, Yinglin Xia, Jun Sun. Vitamin D receptor regulates intestinal barriers in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5215.

LPS-induced Vitamin D Receptor Decrease in Oral Keratinocytes Is Associated With Oral Lichen Planus

The suppressive function of vitamin D on oral lichen planus (OLP) have been documented previously. Vitamin D receptor (VDR) expression is down-regulated in OLP, but the molecular mechanism of its decrease and the related anti-inflammatory contributor of epithelial VDR signaling is unclear. Herein, we demonstrated that lipopolysaccharide (LPS) remarkedly down-regulated VDR expression of keratinocytes, and the reduced regulation was dependent on tumor necrosis factor alpha (TNFα)-miR-346 pathway. In human specimen studies, VDR levels of oral mucosal epithelia from OLP patients decreased substantially accompanied with robust TNFα and miR-346 induction, compared to the normal tissues. In addition, vitamin D/VDR signaling inhibited LPS-induced p53-upregulated modulator of apoptosis (PUMA) induction in keratinocytes via impeding nuclear factor-κB (NF-κB) activation, resulting in keratinocytes apoptosis reduction. Importantly, PUMA activity was up-regulated strongly in diseased epithelium, reversely correlated with VDR expression. Totally, our data indicate that LPS is responsible for VDR downregulation in oral keratinocytes, which is associated with OLP development.

Gut Epithelial Vitamin D Receptor Regulates Microbiota-Dependent Mucosal Inflammation by Suppressing Intestinal Epithelial Cell Apoptosis.

Recent studies show that colonic vitamin D receptor (VDR) signaling protects the mucosal epithelial barrier and suppresses colonic inflammation, but the underlying molecular mechanism remains to be fully understood. To investigate the implication of colonic VDR downregulation seen in patients with inflammatory bowel disease, we assessed the effect of gut epithelial VDR deletion on colonic inflammatory responses in an experimental colitis model. In a 2,4,6-trinitrobenzenesulfonic acid-induced colitis model, mice carrying VDR deletion in gut epithelial cells [VDRflox/flox (VDRf/f);Villin-Cre or VDRΔIEC] or in colonic epithelial cells (VDRf/f;CDX2-Cre or VDRΔCEC) developed more severe clinical colitis than VDRf/f control mice, characterized by more robust T-helper (TH)1 and TH17 responses, with greater increases in mucosal interferon (IFN)-γ+, interleukin (IL)-17+, and IFN-γ+IL-17+ T cells. Accompanying the severe mucosal inflammation was more profound colonic epithelial cell apoptosis in the mutant mice. Treatment with caspase inhibitor Q-VD-OPh dramatically reduced colitis severity and attenuated TH1 and TH17 responses in VDRΔCEC mice. The blockade of cell apoptosis also prevented the increase in mucosal CD11b+CD103+ dendritic cells (DCs), known to be critical for TH17-cell activation. Moreover, depletion of gut commensal bacteria with antibiotics eliminated the robust TH1 and TH17 responses and CD11b+CD103+ DC induction. Taken together, these observations demonstrate that gut epithelial VDR deletion aggravates epithelial cell apoptosis, resulting in increases in mucosal barrier permeability. Consequently, invading luminal bacteria activate CD11b+CD103+ DCs, which promote mucosal TH1 and TH17 responses. Therefore, gut epithelial VDR signaling controls mucosal inflammation by suppressing epithelial cell apoptosis.

Microbiota-Dependent Induction of Colonic Cyp27b1 Is Associated With Colonic Inflammation: Implications of Locally Produced 1,25-Dihydroxyvitamin D3 in Inflammatory Regulation in the Colon.

Our recent studies demonstrated that intestinal epithelial vitamin D receptor (VDR) signaling plays a critical role in regulating colonic inflammation by protecting epithelial barrier integrity. Epithelial VDR is downregulated in colitis, but how mucosal inflammation affects local 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] production is unknown. Here we showed that cytochrome P450 27b1 (Cyp27b1), a cytochrome P450 enzyme necessary for 1,25(OH)2D3 biosynthesis, is highly induced in colonic mucosa in inflammatory response. Although VDR is reduced in colon biopsies from patients with ulcerative colitis, Cyp27b1 is markedly upregulated in these samples. Colon mucosal Cyp27b1 was also markedly induced in an experimental colitis mouse model, and this local Cyp27b1 induction and colonic inflammation required the presence of commensal bacteria. Vitamin D deficiency further exaggerated colonic Cyp27b1 induction and aggravated colonic inflammation in mice. In HCT116 cells, lipopolysaccharide or tumor necrosis factor-α treatment induced Cyp27b1 in time- and dose-dependent manners, and the induced Cyp27b1 was enzymatically active. The inflammation-induced upregulation of Cyp27b1 was mediated by nuclear factor κB. Collectively these data suggest that induction of colonic epithelial Cyp27b1, which is expected to increase local production of 1,25(OH)2D3, is a protective mechanism that partially compensates for the downregulation of epithelial VDR during colonic inflammation. Increased local 1,25(OH)2D3 maintains 1,25(OH)2D3-VDR signaling to protect the mucosal barrier and reduce colonic inflammation.

Vitamin D receptor is a novel transcriptional regulator for Axin1

BackgroundAxin1 is a scaffold protein in the β-catenin destruction complex, which, if disrupted, contributes to pathogenesis of various human diseases, including colorectal carcinogenesis and inflammatory bowel diseases (IBD). We have previously demonstrated that Salmonella infection promotes the degradation and plasma sequestration of Axin1, leading to bacterial invasiveness and inflammatory responses. Vitamin D and the vitamin D receptor (VDR) appear to be important regulators of IBD and colon cancer. Although VDR and Axin1 are all involved in intestinal inflammation, it remains unclear whether these processes are related or function independently. In the current study, we hypothesize that VDR is an important regulator for the maintenance of physiological level of Axin1. MethodsUsing the intestinal epithelial conditional VDR knockout mouse model (VDRΔIEC) and cultured cell lines, influences of VDR status on the expression of Axin1 was evaluated by Western blots and real-time PCR. Loss- and gain-of-function assays were used to investigate the regulation of VDR on Axin1 at the transcriptional and translational levels. Cells were treated with cycloheximide or actinomycin for molecular mechanistic studies. Candidate genomic VDR binding sites for Axin1 were tested by chromatin immunoprecipitation (ChIP) assay. Physical interactions among VDR, Axin1, and β-catenin were tested by immunoprecipitation. Cellular localization of Axin1 with different VDR status was determined by fractionation and immunohistochemistry. ResultsWe found that VDR deletion led to lower protein and mRNA levels of Axin1, whereas knockdown of Axin1 did not change the expression level of VDR protein. Immunoprecipitation data did not support physical interaction between VDR and Axin1. The VDR regulation of Axin1 was through a VDR genomic binding site for Axin1 gene on the regulatory region. Fractionation data showed that cytosolic Axin1 was significantly reduced due to VDR deletion, leaving the nuclear fraction unchanged. In ileum, Axin1 was distributed in the cytosol of apical epithelium and crypts. ConclusionVDR is important for the maintenance of physiological level of Axin1. The discovery of Axin1 as a VDR target gene provides novel and fundamental insights into the interactions between the VDR and β-catenin signaling pathways.

1,25-Dihydroxyvitamin D Protects Intestinal Epithelial Barrier by Regulating the Myosin Light Chain Kinase Signaling Pathway.

The myosin light chain kinase (MLCK) pathway controls intestinal epithelial barrier permeability by regulating the tight junction. 1,25-dihydroxyvitamin D (1,25(OH)2D3)-vitamin D receptor (VDR) signaling protects the epithelial barrier, but the molecular mechanism is incompletely understood. MLCK activation and barrier permeability were studied using monolayers of HCT116, Caco-2, and SW480 cells treated with tissue necrosis factor α with or without 1,25(OH)2D3. The MLCK pathway was analyzed in normal and inflamed colonic biopsies from patients with ulcerative colitis. Colonic mucosal barrier permeability and MLCK activation were also investigated using trinitrobenzene sulfonic acid-induced colitis models in vitamin D analog paricalcitol-treated wild-type mice and mice carrying VDR deletion in colonic epithelial cells. Tissue necrosis factor α increased cell monolayer permeability and induced long isoform of MLCK expression and myosin II regulatory light chain (MLC) phosphorylation, and 1,25(OH)2D3 blocked tissue necrosis factor α-induced increases in monolayer permeability and MLCK-MLC pathway activation by a VDR-dependent fashion. 1,25(OH)2D3 directly suppressed long MLCK expression by attenuating NF-κB activation, and chromatin immunoprecipitation assays confirmed that 1,25(OH)2D3 disrupted p65 binding to 3 κB sites in long MLCK gene promoter. In human ulcerative colitis biopsies, VDR reduction was associated with increases in long MLCK expression and MLC phosphorylation. In trinitrobenzene sulfonic acid colitis models, paricalcitol ameliorated colitis, attenuated the increase in mucosal barrier permeability, and inhibited long MLCK induction and MLC phosphorylation. In contrast, mice with colonic epithelial VDR deletion exhibited more robust increases in mucosal barrier permeability and MLCK activation compared with wild-type mice. These data demonstrate that 1,25(OH)2D3-VDR signaling preserves the mucosal barrier integrity by abrogating MLCK-dependent tight junction dysregulation during colonic inflammation.

Transgenic Expression of Vitamin D Receptor in Gut Epithelial Cells Ameliorates Spontaneous Colitis Caused by Interleukin-10 Deficiency.

Vitamin D deficiency is common in patients with inflammatory bowel diseases. The vitamin D receptor (VDR) is a nuclear hormone receptor mediating the activity of vitamin D hormone. Our previous studies showed that intestinal epithelial VDR signaling inhibits colitis by protecting the mucosal epithelial barrier, and this activity is independent of non-epithelial immune VDR actions. Interleukin (IL)-10-deficient mouse is a chronic colitis model that develops colitis due to aberrant immune responses. Here we used IL-10 null (IL-10KO) model to assess the anti-colitic activity of epithelial VDR in the setting of an aberrant immune system. We crossed IL-10KO mice with villin promoter-driven human (h) VDR transgenic (Tg) mice to generate IL-10KO mice that carry the hVDR transgene in intestinal epithelial cells (IL-10KO/Tg). IL-10KO and IL-10KO/Tg littermates were studied in parallel and followed for up to 25 weeks. By 25 weeks of age, accumulatively 79 % IL-10KO mice developed prolapse, whereas only 40 % IL-10KO/Tg mice did so (P < 0.001). Compared with IL-10KO mice, IL-10KO/Tg littermates showed markedly reduced mucosal inflammation in both small and large intestines, manifested by attenuation in immune cell infiltration and histological damage and a marked decrease in pro-inflammatory cytokine production. IL-10KO/Tg mice also showed reduced intestinal epithelial cell apoptosis as a result of diminished PUMA induction and caspase 3 activation. These observations demonstrate that targeting hVDR expression to intestinal epithelial cells is sufficient to attenuate spontaneous colitis caused by an ill-regulated immune system, confirming a critical role of the epithelial VDR signaling in blocking colitis development.

Intestinal vitamin D receptor protects mice from dysbiosis via modulating JAK/STAT pathway in tumorigenesis

Low vitamin D receptor (VDR) expression and diminished vitamin D/VDR signaling are observed in patients with colon cancer. We reported that intestinal epithelial VDR deletion (VDRΔIEC ) leads to dysbiosis and increases susceptibility to colitis. The JAK/STAT pathway plays a critical role in intestinal and microbial homeostasis. We hypothesized intestinal VDR protects mice from dysbiosis via modulating the JAK/STAT pathway in tumorigenesis. We used an azoxymethane/Dextran sulfate sodium ‐induced cancer model in VDRΔIEC mice. VDRΔIEC mice have higher tumor numbers with tumor location shifted from distal to proximal colon. Fecal microbiota analysis showed that lacking VDR leads to bacterial profile shift from normal to susceptible carcinogenesis. We found enhanced bacterial staining in mouse tumors. These data are consisted with our findings in human tumor sample. VDR deletion decreased Jak2 at protein and mRNA levels. By CHIP assay, we identified that VDR protein bound to the Jak2 promoter, suggesting that VDR transcriptionally regulated Jak2. JAK/STAT was the upstream of the proinflammatory NF‐kappa B pathway. Furthermore, IκBα expression was significantly decreased in VDRΔIEC tumors. Our observations suggest that absence of intestinal epithelial VDR leads to dysbiosis and susceptibility to colon cancer via reducing JAK/STAT signaling and dampening inflammatory responses. Our study provides new insight into the molecular mechanism of VDR regulating the JAK/STAT pathway in bacterial‐host interaction and tumorigenesis. Our studies also indicate a new target—microbiome and VDR signaling for prevention of colon cancer.

Intestinal tight junction CLDN2 gene is a direct target of the vitamin D receptor

The breakdown of intestinal barrier is a common manifestation of GI disorders. Claudin‐2 is a tight junction protein that mediates paracellular water transport in leaky epithelium. Recent evidence suggests that vitamin D and vitamin D receptor (VDR) may regulate functions of tight junction proteins. However, it is unknown how Claudin‐2 is regulated by VDR signaling in normal and inflamed intestine. Using whole body VDR‐/‐ mice, intestinal epithelial VDR conditional knockout (VDRΔIEC) mice, human samples, and cultured human intestinal epithelial cells (IEC), we performed a series of molecular and biochemical experiments in vivo and in vitro. We provide evidence that the CLDN2 gene is a direct target of the transcription factor VDR. VDR‐inducing Claudin‐2 promoter activity required the Cdx binding site on the promoter of the CLDN2 gene. We further identify a functional vitamin D response element in the Claudin‐2 promoter. In vivo, VDR deletion in intestinal epithelial cells (IEC) led to decreased Claudin‐2 at both mRNA and protein levels. Functionally, we found a robust increase of Claudin‐2 in inflammatory IECs which lacked VDR regulation and allowed the inflammatory cytokines to take over. Furthermore, in inflamed intestine of ulcerative colitis patients, VDR expression is low and Claudin‐2 is enhanced. A lack of intestinal VDR regulation leads to dysfunction of Claudin‐2 in inflammatory responses. This study reveals a complex role and novel mechanism for intestinal VDR by regulation of epithelial barriers and inflammation.

Intestinal epithelial vitamin D receptor deletion leads to defective autophagy in colitis

ObjectiveVitamin D and the vitamin D receptor (VDR) appear to be important immunological regulators of inflammatory bowel diseases (IBD). Defective autophagy has also been implicated in IBD, where interestingly, polymorphisms...

Intestinal epithelial vitamin D receptor signaling inhibits experimental colitis

The inhibitory effects of vitamin D on colitis have been previously documented. Global vitamin D receptor (VDR) deletion exaggerates colitis, but the relative anticolitic contribution of epithelial and nonepithelial VDR signaling is unknown. Here, we showed that colonic epithelial VDR expression was substantially reduced in patients with Crohn's disease or ulcerative colitis. Moreover, targeted expression of human VDR (hVDR) in intestinal epithelial cells (IECs) protected mice from developing colitis. In experimental colitis models induced by 2,4,6-trinitrobenzenesulfonic acid, dextran sulfate sodium, or CD4(+)CD45RB(hi) T cell transfer, transgenic mice expressing hVDR in IECs were highly resistant to colitis, as manifested by marked reductions in clinical colitis scores, colonic histological damage, and colonic inflammation compared with WT mice. Reconstitution of Vdr-deficient IECs with the hVDR transgene completely rescued Vdr-null mice from severe colitis and death, even though the mice still maintained a hyperresponsive Vdr-deficient immune system. Mechanistically, VDR signaling attenuated PUMA induction in IECs by blocking NF-κB activation, leading to a reduction in IEC apoptosis. Together, these results demonstrate that gut epithelial VDR signaling inhibits colitis by protecting the mucosal epithelial barrier, and this anticolitic activity is independent of nonepithelial immune VDR actions.