Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which often leads to protein misfolding and no CFTR surface localization. This then leads to chronic airway infections, inflammation, and tissue damage. Although vitamin D has been explored as a therapy to treat CF due to its antimicrobial-inducing and anti-inflammatory properties, the effect of 1,25-dihydroxyvitamin D3 (1α,25(OH)2D3) on CFTR directly has not been studied. We treated cultured healthy and diseased bronchial epithelial cells (BEC) with 10nM 1α,25(OH)2D3 for 6 and 24h and found that 1α,25(OH)2D3 increases both mRNA and protein CFTR levels using RT-qPCR, flow cytometry and fluorescence immunohistochemistry. Treatment of CF cells with 10nM 1α,25(OH)2D3 led to an increase in both total and surface CFTR expression, suggesting 1α,25(OH)2D3 could be used to increase properly localized CFTR in airway cells. To determine if BEC could convert the more clinically relevant cholecalciferol to 25OHD3, cultured non-CF and CF BECs were treated with a range of cholecalciferol concentrations, and 25OHD3 levels were quantified by ELISA. We found that 25OHD3 levels increased in a concentration-dependent manner. Treatment of BEC with 10μM cholecalciferol led to increases in both CYP24A1 and CFTR mRNA levels, even when added to the apical surface of cells grown in an air-liquid interface, suggesting that topical administration of vitamin D could be used therapeutically. To demonstrate this in vivo, we intranasally delivered 1μM 1α,25(OH)2D3 into mice. After 6h, we observed induction of both Cyp24A1 and CFTR expression in the tracheas of treated mice. The major findings of this study are that vitamin D can be converted to the active form when topically administered to the airway, and this could be used to increase CFTR levels in patients with CF. This could potentially be useful as an adjunctive therapy, together with newly developed CF treatments.

The vitamin D-modulated transcriptome of highly responsive human cells, such as THP-1 monocytes, comprises more than 500 genes, half of which are primary targets. Recently, we proposed a chromatin model of vitamin D signaling demonstrating that nearly all vitamin D target genes are located within vitamin D-modulated topologically associated domains (TADs). This model is based on genome-wide binding patterns of the vitamin D receptor (VDR), the pioneer transcription factor PU.1, the chromatin organizer CTCF and histone markers of active promoter regions (H3K4me3) and active chromatin (H3K27ac). In addition, time-dependent data on accessible chromatin and mRNA expression are implemented. For the interrogation and in deep inspection of these high-dimensional datasets unsupervised and supervised machine learning algorithms were applied. Unsupervised methods, such as the vector quantization tool K-means and the dimensionality reduction algorithm self-organizing map, generated descriptions of how attributes, such as VDR binding and chromatin accessibility, affect each other as a function of time and/or co-localization within the same genomic region. Supervised algorithms, such as random forests, allowed the data to be classified into pre-existing categories like persistent (i.e. constant) and time-dependent (i.e. transient) VDR binding sites. The relative amounts of these VDR categories in TADs showed to be the main discriminator for sorting the latter into five classes carrying vitamin D target genes involved in distinct biological processes. In conclusion, via the application of machine learning methods we identified the spatio-temporal VDR binding pattern in TADs as the most critical attribute for specific regulation of vitamin D target genes and the segregation of vitamin D's physiologic function.

Melanoma is the most dangerous form of skin cancer, with poor prognosis in advanced stages. Vitamin D, also produced by ultraviolet radiation, is known for its anti-proliferative properties in some cancers including melanoma. While vitamin D deficiency has been associated with advanced melanoma stage and higher levels of vitamin D have been associated with better outcomes, the role for vitamin D in melanoma remains unclear. Vitamin D synthesis is initiated upon UVB exposure of skin cells and results in formation of the active metabolite 1,25-dihydroxyvitamin D3 (1,25D). We have previously demonstrated that 1,25D plays a role in protection against ultraviolet radiation-induced DNA damage, immune suppression, and skin carcinogenesis. In this study 1,25D significantly reduced cell viability and increased caspase levels in human melanoma cell lines. This effect was not present in cells that lacked both phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a well-known tumour suppressor, and the vitamin D receptor (VDR). PTEN is frequently lost or mutated in melanoma. Incubation of selected melanoma cell lines with 1,25D resulted in significant increases in PTEN levels and downregulation of the AKT pathway and its downstream effectors. This suggests that 1,25D may act to reduce melanoma cell viability by targeting PTEN.

The vitamin D system is deregulated during development and progression of several cancer types. Data on the expression of the vitamin D system in the diseased pancreas are missing. The aim of this study was to investigate the expression of the vitamin D receptor (VDR), 1,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1), and the calcium-sensing receptor (CaSR), a vitamin D target gene, in the different regions of the pancreas in patients with chronic pancreatitis (n=6) and pancreatic ductal adenocarcinomas (PDAC) (n=17). We analyzed the expression of these genes at mRNA and protein level with quantitative real-time RT-PCR and immunostaining. mRNA expression of CYP24A1 and VDR was significantly increased in tumors compared with the adjacent non-tumorous tissue (p<0.01), while CaSR mRNA expression decreased. Both the VDR and the CaSR protein were highly expressed in the endocrine compared with the exocrine pancreas. In CP the CYP24A1 expression was highest in the endocrine pancreas, while in PDACs in the transformed ducts. In the PDAC patients CYP24A1 expression in the islets was significantly lower than in CP patients. Our data suggest that during ductal adenocarcinoma development the vitamin D system in the pancreas becomes deregulated on two levels: in the islets CYP24A1 expression decreases weakening the negative feedback regulation of the vitamin D-dependent insulin synthesis/secretion. In the transformed ducts CYP24A1 expression increases, impairing the antiproliferative effect of vitamin D in these cells.

Vitamin D (VD) is essential for the human body and involved in a wide variety of critical physiological processes including bone, muscle, and cardiovascular health, as well as innate immunity and antimicrobial responses. Here, we elucidated the significance of the VD system in cytomegalovirus (CMV) infection, which is one of the most common opportunistic infections in immunocompromised or -suppressed patients. We found that expression of vitamin D receptor (VDR) was downregulated in CMV-infected cells within 12h [hrs] post infection [p.i.] to 12% relative to VDR expression in mock-infected fibroblasts and did not recover during the CMV replication cycle of 96h. None of the biologically active metabolites of VD, cholecalciferol, calcidiol, or calcitriol, inhibit CMV replication significantly in human fibroblasts. In a feedback loop, expression of CYP24A1 dropped to 3% by 12h p.i. and expression of CYP27B1 increased gradually during the replication cycle of CMV to 970% probably as a consequence of VDR inhibition. VDR expression was not downregulated during influenza virus or adenovirus replication. The potent synthetic vitamin D analog EB-1089 was not able to inhibit CMV replication or antagonize its effect on VDR expression. Only CMV replication, and none of the other viral pathogens evaluated, inhibited the vitamin D system in vitro. In view of the pleiotropism of VDR, CMV-mediated downregulation may have far-reaching virological, immunological, and clinical implications and thus warrant further evaluations in vitro and in vivo.

Vitamin D3 is a pleiotropic signaling molecule that has via activation of the transcription factor vitamin D receptor (VDR) a direct effect on the expression of more than 100 genes. The aim of this study was to find transcriptomic and clinical biomarkers that are most suited to identify vitamin D3 responders within 71 pre-diabetic subjects during a 5-month intervention study (VitDmet). In hematopoietic cells, the genes ASAP2, CAMP, CD14, CD97, DUSP10, G0S2, IL8, LRRC8A, NINJ1, NRIP1, SLC37A2 and THBD are known as primary vitamin D targets. We demonstrate that each of these 12 genes carries a conserved VDR binding site within its genomic region and is expressed in human peripheral blood mononuclear cells (PBMCs). The changes in the expression of these genes in human PBMCs at the start and the end of the vitamin D-intervention were systematically correlated with the alteration in the circulating form of vitamin D3, 25-hydroxyvitamin D3 (25(OH)D3). Only 39-44 (55-62%) of the study subjects showed a highly significant response to vitamin D3, i.e., we considered them as "responders". In comparison, we found for 37-53 (52-75%) of the participants that only 12 biochemical and clinical parameters, such as concentrations of parathyroid hormone (PTH) and insulin, or computed values, such as homeostatic model assessment and insulin sensitivity index, show a correlation with serum 25(OH)D3 levels that is as high as that of the selected VDR target genes. All 24 parameters together described the pleiotropic vitamin D response of the VitDmet study subjects. Interestingly, they demonstrated a number of additional correlations that define a network, in which PTH plays the central role. In conclusion, vitamin D3-induced changes in human PBMCs can be described by transcriptomic and serum biomarkers and allow a segregation into high and low responders. This article is part of a Special Issue entitled '17th Vitamin D Workshop' .

Accumulating evidence has proposed a correlation between vitamin D (25(OH)D) insufficiency and cardiovascular (CV) disease. Vitamin D associated effects on endothelial function have been suggested to be a possible culprit. The present study investigated the association of vitamin D3 treatment on markers of endothelial dysfunction in patients with arterial hypertension.

In humans and other primates, 1,25(OH)2vitamin D3 regulates the expression of the cathelicidin antimicrobial peptide (CAMP) gene via toll-like receptor (TLR) signaling that activates the vitamin D pathway. Mice and other mammals lack the vitamin D response element (VDRE) in their CAMP promoters. To elucidate the biological importance of this pathway, we generated transgenic mice that carry a genomic DNA fragment encompassing the entire human CAMP gene and crossed them with Camp knockout (KO) mice. We observed expression of the human transgene in various tissues and innate immune cells. However, in mouse CAMP transgenic macrophages, TLR activation in the presence of 25(OH)D3 did not induce expression of either CAMP or CYP27B1 as would normally occur in human macrophages, reinforcing important species differences in the actions of vitamin D. Transgenic mice did show increased resistance to colonization by Salmonella typhimurium in the gut. Furthermore, the human CAMP gene restored wound healing in the skin of Camp KO mice. Topical application of 1,25(OH)2vitamin D3 to the skin of CAMP transgenic mice induced CAMP expression and increased killing of Staphylococcus aureus in a wound infection model. Our model can help elucidate the biological importance of the vitamin D-cathelicidin pathway in both pathogenic and non-pathogenic states.

We aimed to evaluate sex differences in changes of lipid profiles in a cohort of metabolically healthy adults following Orthodox fasting (OF), as well as to assess a potential role of vitamin D status in mediating these variations. 45 individuals (24 premenopausal females, 53.3 %) with mean age 48.3 ± 9.1 years and mean Body Mass Index 28.7 ± 5.8 kg/m2 were prospectively followed for 12 weeks. Anthropometry, dietary and biochemical data regarding serum lipids, and vitamin D status were collected at baseline, 7 weeks after the implementation of OF, and 5 weeks after fasters returned to their standard dietary habits (12 weeks from baseline). According to 25-hydroxy-vitamin D [25(OH)D] measurements, participants were divided into two groups: those with concentrations above and below the median of values. Females with 25(OH)D concentrations below the median manifested a non-significant reduction by approximately 15 % in total and low-density lipoprotein cholesterol during the fasting period, followed by a significant increase 5 weeks after OF cessation (170.7 vs. 197.5 and 99.6 vs. 121.0 mg/dl respectively, p < 0.001). In contrast, males with 25(OH)D levels below the median demonstrated an inverse, non-significant trend of increase in lipid concentrations during the whole study period. Our findings suggest strikingly different inter-gender lipid responses to a dietary model of low-fat, mediated by vitamin D status. Further studies are necessary to reveal the underlying mechanisms and assess the importance of these differences with respect to cardiovascular health and the benefit of vitamin D supplementation strategies.

Multiple Sclerosis (MS) is often associated with low serum 25(OH)D levels, as well as cognitive dysfunctions. The relationship between 25(OH)D and the most commonly affected cognitive domain in MS; processing speed, is poorly explored. The purpose of this study is to: (1) assess the effect of serum 25(OH)D change on processing speed in MS, and (2) explore the relationship between serum 25(OH)D and brain volume changes in MS. A retrospective chart review was conducted, data from 299 patients were extracted (baseline), of whom 163 had follow-up measurements (after at least a 9-month interval). The Symbol Digits Modalities Test (SDMT) was used as a measure of processing speed. MRI data was available from 78 individuals at baseline, and 70 at follow-up. SDMT scores and brain volumes (Cerebellum (total, grey, and white), intracranial, Grey Matter (GM), and White Matter (WM)) were compared based on 25(OH)D levels and their changes towards follow-up. Results indicated that patients with deficient 25(OH)D levels had lower SDMT scores when compared to those with sufficient levels, and SDMT scores improved as a function of 25(OH)D. For MRI measures, only patients with sufficient 25(OH)D levels during both assessment periods had significant changes in intracranial and total cerebellum volumes. We conclude that 25(OH)D levels seem to have an effect on processing speed in MS, thus the importance of clinical monitoring and supplementation in this regard is reinforced.

Chronic obstructive pulmonary disease (COPD), which is characterized by an excessive inflammatory response of the airways, is often complicated by exacerbations. Vitamin D deficiency has been associated with an increased risk for COPD and may predispose COPD patients to a higher exacerbation rate, particularly during smoking. In the current study, we investigated the effect of vitamin D deficiency and cigarette smoke (CS)-exposure on lung inflammation and bacterial clearance after an acute infection with Nontypeable Haemophilus influenzae (NTHi). Vitamin D deficient or sufficient mice were exposed to nose-only CS or ambient air for 6 weeks and oropharyngeally instilled with 106 NTHi. Residual viable NTHi were measured at different time points post-infection. Mechanisms of bacterial clearance (e.g. phagocytosis, pattern recognition receptors, antimicrobial peptides, surfactant proteins and mucin) and lung remodeling (e.g. metalloproteinases, MMP's) were assessed. Although smoking resulted in reduced phagocytosis capacity of macrophages and neutrophils, bacterial clearance was similar to control mice. By contrast and independent of smoking, bacterial clearance was significantly accelerated in vitamin D deficient mice already from 24 h post-infection (p = 0.0087). This faster and complete eradication was associated with a more rapid resolution of cytokines and neutrophils 72 h post-infection and dominated by an upregulation of cathelicidin-related antimicrobial peptide (CRAMP) mRNA during infection (p = 0.026). However, vitamin D deficiency also resulted in more MMP12 protein in broncho-alveolar lavage and a shift in mRNA expression of MMP12/TIMP1 (p = 0.038) and MMP9/TIMP1 (p = 0.024) ratio towards more protease activity. Overall, vitamin D deficient mice resolved NTHi infection faster with a faster resolution of local lung inflammation, possibly through upregulation of CRAMP. This was associated with a disruption of the protease/anti-protease balance, which may potentially scale towards a higher extracellular matrix breakdown.

Vitamin D(3) metabolites regulate the bone metabolism and 1α,25-dihydroxyvitamin D(3) (1α,25(OH)(2)D(3)) is known to play an important role in teeth mineralization. However, little is known about the potential of vitamin D as an osteogenic inducer in human dental pulp (hDPCs) and dental follicle cells (hDFCs) in vitro. Therefore, we investigated the effects of vitamin D(3) metabolites 1α,25(OH)(2)D(3) and 25-hydroxyvitamin D(3) (25OHD(3)) on proliferation and osteogenic differentiation of hDPCs and hDFCs in vitro. We also examined whether vitamin D(3) metabolic enzymes were regulated in hDFCs and hDPCs. Cell proliferation was decreased by both metabolites in hDPCs and hDFCs. Vitamin D(3) metabolites increased ALP activity and induced mineralization when osteogenic supplements (OS; l-ascorbic acid-2-phosphate+β-glycerophosphate) were added, though the expression of osteocalcin (OC) and osteopontin (OPN) were regulated without the addition of OS. CYP24 and CYP27B1 expressions were upregulated by vitamin D(3) metabolites and 25OHD(3) was converted into 1α,25(OH)(2)D(3) in the culture medium. These results confirm that 1α,25(OH)(2)D(3) (10 and 100 nM) and 25OHD(3) (500 nM) can be used as osteogenic inducers synergistically with osteogenic supplements for differentiation of hDPCs and hDFCs. Furthermore, our findings strengthen our knowledge about the role of hDPCs and hDFCs as vitamin D(3) target cells.

The arachidonate 5-lipoxygenase (ALOX5) pathway has been implicated in chronic inflammatory disease which may be influenced by vitamin D due to vitamin D response elements (VDRE). We investigated an ALOX5 polymorphism (rs4987105) in patients with type 2 diabetes (T2D) and the in vitro effects of calcitriol (1,25(OH)2D3) on ALOX5 metabolism in monocytes of T2D patients and healthy controls (HC). 533 T2D and 473 HC were genotyped for the rs4987105 polymorphism. In addition, the 25(OH)D3 and 1,25(OH)2D3 plasma levels were measured in both cohorts. Further C-reactive protein (CRP) was determined in T2D patients. Our results demonstrate, that genotype CC and the allele C of ALOX5 rs4987105 polymorphism were more frequent in T2D compared to HC (OR = 1.44; 95% CI: 1.12-1.84; p < 0.05). Lower levels of both vitamin D metabolites (p < 0.0001 respectively) were found in the CC genotyped T2D patients compared to CC genotyped HC. In addition, CC genotyped T2D patients had higher levels of CRP compared to CT and TT genotyped T2D patients, (p < 0.01). In order to evaluate the impact of calcitriol in primary isolated monocytes, we isolated monocytes of 20 T2D patients and 20 HC. The cells were treated with 1,25(OH)2D3 and interleukin-1beta (IL-1β) for 24 h. The following genes were analysed for expression changes: ALOX5, leukotriene A4 hydrolase (LTA4H), leukotriene B4 receptor type 1 (LTB4R1) and CD14. Treatment with IL-1β+1,25(OH)2D3 increased ALOX5, LTA4H and LTB4R1 and CD14 mRNA in both T2D patients and HC (p < 0.0001, respectively). In addition, IL-1β+1,25(OH)2D3 treatment led to higher ALOX5, LTA4H and CD14 mRNA levels in T2D patients compared to HC (p < 0.001, p < 0.05, p ≤ 0.05, respectively). In conclusion, ALOX5 rs4987105 allele C confers susceptibility to T2D, lower vitamin D metabolites and higher CRP levels complement this association. Additionally, IL-1β+1,25(OH)2D3 treatment on, ALOX5, LTA4H and CD14 mRNA indicate a diabetes specific modulation. These findings identify a novel pathway in T2D potentially amenable for individualized therapeutic targeting.

A large number of human, animal and in vitro studies have suggested that vitamin D3 (VD3) plays a critical role in inflammatory airway diseases such as asthma, chronic rhinosinusitis, and allergic rhinitis. VD3 acts upon a broad range of immune cells involved in the pathogenesis of these diseases including T-cells, dendritic cells (DCs), macrophages, and B-cells. In addition, VD3 can also regulate the functions of a number of non-immune cells including epithelial cells, fibroblasts, and smooth muscle cells. Given that VD3 has known effects on the immune system, it seems logical that supplementation with VD3 would prove efficacious in the treatment of these three diseases. While many studies, most of which are observational, have suggested that VD3 deficiency is associated with more severe disease, VD3 supplementation trials in humans have resulted in varied outcomes in terms of efficacy. In this review article we will discuss the role of VD3 in these three commonly associated respiratory diseases. We will explore the literature describing associations of VD3 deficiency with patient outcomes, cells in the respiratory microenvironment susceptible to VD3 regulation, conflicting results of VD3 supplementation trials, and potential gaps in our knowledge that may be limiting the widespread use of VD3 for the treatment of respiratory diseases such asthma, chronic rhinosinusitis and allergic rhinitis. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

The active form of Vitamin D (1,25(OH)2D), has been suggested to have a regulatory role in skeletal muscle function and metabolism, however, the effects and mechanisms of vitamin D (VitD) action in this tissue remain to be fully established. In this study, we have used primary human skeletal muscle myoblast (HSMM) cells that display typical characteristics of human skeletal muscle function and protein levels, to investigate the effects of the active form of VitD on proliferation, differentiation, protein synthesis and bioenergetics. Myoblast cells were treated with 100 nM of VitD for 24 h, 48 h, 72 h and five days (cells were differentiated into myotubes) and then analyses were performed. We report that VitD inhibits myoblast proliferation and enhances differentiation by altering the expression of myogenic regulatory factors. In addition, we found that protein synthesis signaling improved in myotubes after VitD treatment in the presence of insulin. We also report an increase in oxygen consumption rate after 24 h of treatment in myoblasts and after 5 days of treatment in myotubes after VitD exposure. VitD significantly impacted HSMM myogenesis, as well as protein synthesis in the presence of insulin.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer and it is the third leading cause of global cancer mortality. Sorafenib (Sf) is the first oral multi-kinase inhibitor approved for systemic treatment of advanced HCC, and can prolong survival, although only for three months longer than placebo treated patients. Preclinical studies showed that active forms of vitamin D can induce cell differentiation and regulate cell survival in several cell types, and epidemiological data link vitamin D insufficiency to an increased risk of neoplastic diseases, suggesting a potentially important role of vitamin D in cancer therapy. Other studies showed that the effect of vitamin D analogs on human neoplastic cells is potentiated by carnosic acid (CA), a plant polyphenol with anti-oxidant properties. Here we tested if the addition of the vitamin D2 analog Doxercalciferol (D2) together with CA can enhance the cytotoxic effect of Sf on HCC cell lines Huh7 (Sf-sensitive) and HCO2 (Sf-resistant). Indeed, this combination increased HCC cell death in cell lines, enhancing autophagy as well as apoptosis. Autophagy was confirmed by increased cytoplasmic vacuolation, perinuclear aggregation of LC3, and elevated protein levels of autophagy markers Beclin1, Atg3, and LC3. These results suggest that a regimen which combines a vitamin D2 analog/CA mixture with Sf can be a novel and promising therapeutic option for the treatment of HCC.

Transfer and metabolism of vitamin D across the human placenta is required for fetal development. However, these fundamental mechanisms are not well understood and model systems are required to help understand them. The BeWo choriocarcinoma cell line is derived from extravillous trophoblast but is used as a model for villous syncytiotrophoblast and the placental barrier. Questions have been raised about the suitability of the BeWo cell line as a model for villous trophoblast. This study compares the expression of amino acid transporters and vitamin D related genes in human term placenta with the BeWo and human embryonic kidney (HEK)293 cell lines. HEK293 cells, as transporting epithelium may be more similar to placenta. Gene expression in term placenta was much more similar to HEK293 than BeWo. This study provides further evidence that the BeWo cell line is not an appropriate model for villous trophoblast and a model that more closely represents the human placenta is now required to investigate the effects of vitamin D on the placenta ex-vivo.

Accumulating evidence suggests a potential implication of vitamin D biological network in the pathogenesis of diabetes mellitus. The megalin-cubilin endocytotic system constitutes a key transport structure, with a modulating role in vitamin D metabolism. We aimed to assess the contribution of variants in the CUBN gene to the genetic risk of Type 2 Diabetes Mellitus (T2DM). 95 polymorphisms within CUBN were genotyped in 716 patients with T2DM and 542 controls of Greek origin. Samples were analyzed on Illumina Human PsychArray. Permutation test analysis was implemented to determine statistical significance. Twenty-five-hydroxy-vitamin-D [25(OH)D)] levels were measured in a sub-group of participants (n = 276). Permutation analysis associated rs11254375_G/T (pemp = 0.00049, OR = 1.482), rs6602175_G/T (pemp = 0.016, OR = 0.822), rs1801224_G/T (pemp = 0.025, OR = 0.830), rs4366393_A/G (pemp = 0.028, OR = 0.829) and rs7071576_A/G (pemp = 0.04, OR = 1.219) with T2DM. Mean 25(OH)D concentrations were significantly lower in patients with T2DM compared to controls (16.70 ± 6.69 ng/ml vs 18.51 ± 6.71 ng/ml, p < 0.001), although both groups were vitamin D deficient. In a further quantitative analysis, rs41301097 was strongly associated with higher 25(OH)D concentrations (p = 5.233e-6, beta = 15.95). Our results indicate a potential role of CUBN gene in T2DM genetic susceptibility in the Greek population. These findings may also denote an indirect effect of vitamin D metabolism dysregulation on the pathogenesis of T2DM. Further studies are required to replicate our findings and clarify the complex underlying mechanisms.

While many studies have addressed the direct effects of 1alpha,25(OH)2D3 on breast cancer (BC) cells, stromal-epithelial interactions, which are important for the tumor development, have been largely ignored. In addition, high concentrations of the hormone, which cannot be attained in vivo, have been used. Our aim was to establish a more physiological breast cancer model, represented by BC tissue slices, which maintain epithelial-mesenchymal interactions, cultured with a relatively low 1alpha,25(OH)2D3 concentration, in order to evaluate the vitamin D pathway. Freshly excised human BC samples were sliced and cultured in complete culture media containing vehicle, 0.5 nM or 100 nM 1alpha,25(OH)2D3 for 24 h. BC slices remained viable for at least 24 h, as evaluated by preserved tissue morphology in hematoxylin and eosin (HE) stained sections and bromodeoxyuridine (BrdU) incorporation by 10% of tumor cells. VDR mRNA expression was detected in all samples and CYP24A1 mRNA expression was induced by 1alpha,25(OH)2D3 in both concentrations (but mainly with 100 nM). Our results indicate that the vitamin D signaling pathway is functional in BC slices, a model which preserves stromal-epithelial interactions and mimics in vivo conditions.

Despite correlations between low vitamin D levels and diabetes incidence/severity, supplementation with vitamin D has not been widely effective in improving glucose parameters. This may be due to a lack of knowledge regarding how low vitamin D levels physiologically affect glucose homeostasis. We have previously shown that the brain may be a critical area for vitamin d-mediated action on peripheral glucose levels. However, the mechanisms for how vitamin D acts in the brain are unknown. We utilized a multimodal approach to determine the mechanisms by which vitamin D may act in the brain. We first performed an unbiased search (RNA-sequencing) for pathways affected by vitamin D. Vitamin D (125-dihydroxyvitamin D3; 1,25D3) delivered directly into the third ventricle of obese animals differentially regulated multiple pathways, including the insulin signaling pathway. The insulin signaling pathway includes PI3K, which is important in the brain for glucose regulation. Since others have shown that vitamin D acts through the PI3K pathway in non-neuronal cells (muscle and bone), we hypothesized that vitamin D may act in neurons through a PI3K-dependent pathway. In a hypothalamic cell-culture model (GT1-7 cells), we demonstrate that 1,25D3 increased phosphorylation of Akt in the presence of insulin. However, this was blocked with pre-treatment of wortmannin, a PI3K inhibitor. 1,25D3 increased gene transcription of several genes within the PI3K pathway, including Irs2 and p85, without affecting expression of InsR or Akt. Since we had previously shown that 1,25D3 has significant effects on neuronal function, we also tested if the PI3K pathway could mediate rapid actions of vitamin D. We found that 1,25D3 increased the firing frequency of neurons through a PI3K-dependent mechanism. Collectively, these data support that vitamin D enhances insulin signaling and neuronal excitability through PI3K dependent processes which involve both transcriptional and membrane-initiated signaling events.