Vitamin D receptor (VDR) executes most of the biological functions of vitamin D. Beyond this, VDR is a transcriptional factor regulating the expression levels of many target genes, such as genes for tight junction proteins claudin-2, -5, -12, and -15. In this review, we discuss the progress of research on VDR that influences intestinal barriers in health and disease. We searched PubMed and Google Scholar using key words vitamin D, VDR, tight junctions, cancer, inflammation, and infection. We summarize the literature and progress reports on VDR regulation of tight junction distribution, cellular functions, and mechanisms (directly or indirectly). We review the impacts of VDR on barriers in various diseases, e.g., colon cancer, infection, inflammatory bowel disease, and chronic inflammatory lung diseases. We also discuss the limits of current studies and future directions. Deeper understanding of the mechanisms by which the VDR signaling regulates intestinal barrier functions allow us to develop efficient and effective therapeutic strategies based on levels of tight junction proteins and vitamin D/VDR statuses for human diseases.

Background: Transfer RNA (tRNA) queuosine (Q)-modifications occur specifically in 4 cellular tRNAs at the wobble anticodon position. tRNA Q-modification in human cells depends on the gut microbiome because the microbiome product queuine is required for its installation by the enzyme Q tRNA ribosyltransferase catalytic subunit 1 (QTRT1) encoded in the human genome. Queuine is a micronutrient from diet and microbiome. Although tRNA Q-modification has been studied for a long time regarding its properties in decoding and tRNA fragment generation, how QTRT1 affects tumorigenesis and the microbiome is still poorly understood. Results: We generated single clones of QTRT1-knockout breast cancer MCF7 cells using Double Nickase Plasmid. We also established a QTRT1-knockdown breast MDA-MB-231 cell line. The impacts of QTRT1 deletion or reduction on cell proliferation and migration in vitro were evaluated using cell culture, while the regulations on tumor growth in vivo were evaluated using a xenograft BALB/c nude mouse model. We found that QTRT1 deficiency in human breast cancer cells could change the functions of regulation genes, which are critical in cell proliferation, tight junction formation, and migration in human breast cancer cells in vitro and a breast tumor mouse model in vivo. We identified that several core bacteria, such as Lachnospiraceae, Lactobacillus, and Alistipes, were markedly changed in mice post injection with breast cancer cells. The relative abundance of bacteria in tumors induced from wildtype cells was significantly higher than those of QTRT1 deficiency cells. Conclusions: Our results demonstrate that the QTRT1 gene and tRNA Q-modification altered cell proliferation, junctions, and microbiome in tumors and the intestine, thus playing a critical role in breast cancer development.

Microbial metabolites affect the neuron system and muscle cell functions. Amyotrophic lateral sclerosis (ALS) is a multifactorial neuromuscular disease. Our previous study has demonstrated elevated intestinal inflammation and dysfunction of the microbiome in patients with ALS and an ALS mouse model (human-SOD1^G93A transgenic mice). However, the metabolites in ALS progression are unknown. Using an unbiased global metabolomic measurement and targeted measurement, we investigated the longitudinal changes of fecal metabolites in SOD1^G93A mice over the course of 13 weeks. We further compared the changes of metabolites and inflammatory response in age-matched wild-type (WT) and SOD1^G93A mice treated with the bacterial product butyrate. We found changes in carbohydrate levels, amino acid metabolism, and the formation of gamma-glutamyl amino acids. Shifts in several microbially contributed catabolites of aromatic amino acids agree with butyrate-induced changes in the composition of the gut microbiome. Declines in gamma-glutamyl amino acids in feces may stem from differential expression of gamma-glutamyltransferase (GGT) in response to butyrate administration. Due to the signaling nature of amino acid-derived metabolites, these changes indicate changes in inflammation, e.g., histamine, and contribute to differences in systemic levels of neurotransmitters, e.g., γ-Aminobutyric acid (GABA) and glutamate. Butyrate treatment was able to restore some of the healthy metabolites in ALS mice. Moreover, microglia in the spinal cord were measured by IBA1 staining. Butyrate treatment significantly suppressed the IBA1 level in the SOD1^G93A mice. Serum IL-17 and LPS were significantly reduced in the butyrate-treated SOD1^G93A mice. We have demonstrated an inter-organ communications link among microbial metabolites, neuroactive metabolites from the gut, and inflammation in ALS progression. The study supports the potential to use metabolites as ALS hallmarks and for treatment.

Sorting Nexin 27 (SNX27) belongs to a family of sortin nexins and possesses a unique binding domain at the C-terminus which mediates protein-protein interaction in intracellular trafficking, membrane remodeling, organelle motility, and tight junctions. However, its role in cancer development, especially in vivo, remains largely unknown. We have generated a stable SNX27 knockdown clone in a highly aggressive breast cancer cell line MDA-MB-231 using an inducible lentiviral shRNA system. Cell migration and proliferation of SNX27 knockdown (KD) cells were compared with wild-type (WT) cells by MTT and wound healing assay, respectively. The differences in colony formation between SNX27-KD and WT cells were detected by soft agar culture and matrigel 3D culture. Furthermore, tumor growth was examined in a xenograft nude mouse model using SNX27-KD and WT MDA-MB-231 cells. The critical EMT (epithelial-mesenchymal transition) regulators were examined in vitro and in vivo. The wound healing assay showed that SNX27 knockdown significantly decreased cell motility and proliferation. Colony formation in soft agar showed that the SNX27 knockdown cells formed significantly fewer and smaller colonies than the parental MDA-MB-231 cells. Western blots and immunostaining showed that knockdown of SNX27 led to increased expression of E-cadherin and β-catenin proteins, which facilitate adhesion formation and reverse EMT. EMT is a cellular program that allows polarized, immotile epithelial cells to convert to motile mesenchymal cells, promoting carcinoma invasion. The expression levels of Vimentin, the transcription factor of EMT, and tight junction protein Claudin-5, were significantly diminished in the SNX27 knockdown cells. The expression of PCNA, the cell proliferation marker, was increased in SNX27-KD cells transfected with E-cadherin siRNA. In a xenograft nude mouse model, we found that knockdown of SNX27 significantly inhibited tumor growth. The tumors from mice with SNX27-KD cells showed less proliferation compared to tumors from mice injected with wildtype cells. The increase in E-cadherin and β-catenin and decrease in Vimentin and Claudin-5 were observed in tumors of mice injected with SNX27-KD cells. Our data have demonstrated that SNX27 plays a crucial role in tumor growth in vitro and in vivo.

Radiation damage is associated with inflammation and immunity in the intestinal mucosa, including gut microbiota. Melanin has a unique capacity to coordinate a biological reaction in response to environmental stimuli, such as radiation exposure. Thus, melanin and melanized microbes have potential to be used for mitigation of injury induced by radiation. The purpose of the current study is to examine the safety of these agents for future targeting gut microbiome to prevent radiation-induced injury. We administered mice with soluble allomelanin and observed its effect on the intestinal physiology and body weight. We then established a melanized bacterial strain in probiotic E. coli Nissle. We measured the body weight of the mice treated with melanized E. coli Nissle. We showed the enhanced bacterial abundance and colonization of the melanized bacteria E. coli Nissle in the intestine. Melanized E. coli Nissle colonized the colon in less than 3 h and showed consistent colonization over 24 h post one oral gavage. We did not find significant changes of bodyweight in the mice treated with melanized bacteria. We did not observe any inflammation in the intestine. These results demonstrate the safety of soluble melanin and melanin-producing bacteria and will support the future studies to treat radiation-induced injuries and restore dysbiosis.

Vitamin D signaling via the Vitamin D Receptor (VDR) has been shown to protect against intestinal inflammation. Previous studies have also reported the mutual interactions of intestinal VDR and the microbiome, indicating a potential role of probiotics in modulating VDR expression. In preterm infants, although probiotics have been shown to reduce the incidence of necrotizing enterocolitis (NEC), they are not currently recommended by the FDA due to potential risks in this population. No previous studies have delved into the effect of maternally administered probiotics on intestinal VDR expression in early life. Using an infancy mouse model, we found that young mice exposed to maternally administered probiotics (SPF/LB) maintained higher colonic VDR expression than our unexposed mice (SPF) in the face of a systemic inflammatory stimulus. These findings indicate a potential role for microbiome-modulating therapies in preventing diseases such as NEC through the enhancement of VDR signaling.