Vitamin D Receptor mRNA Research Articles

The role of 25-hydroxyvitamin-D3 and vitamin D receptor gene in human periodontal ligament fibroblasts as response to orthodontic compressive strain: an in vitro study

BackgroundThis study aimed to investigate, if different physiological concentrations of vitamin D (25(OH)D3) and single nucleotide polymorphisms in vitamin D receptor (VDR) gene have an impact on gene expression in human periodontal ligament (hPDL) fibroblasts induced by simulated orthodontic compressive strain.MethodsA pool of hPDL fibroblasts was treated in absence or presence of 25(OH)D3 in 3 different concentrations (10, 40 and 60 ng/ml). In order to evaluate the role of single nucleotide polymorphisms in the VDR gene, hPDL fibroblasts from 9 patients were used and treated in absence or presence of 40 ng/ml 25(OH)D3. Each experiment was performed with and without simulated orthodontic compressive strain. Real-time PCR was used for gene expression and allelic discrimination analysis. Relative expression of dehydrocholesterol reductase (DHCR7), Sec23 homolog A, amidohydrolase domain containing 1 (AMDHD1), vitamin D 25-hydroxylase (CYP2R1), Hydroxyvitamin D-1-α hydroxylase, receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), cyclooxygenase-2 (COX-2) and interleukin-6 (IL6) was assessed. Three single nucleotide polymorphisms in VDR were genotyped. Parametric or non-parametric tests were used with an alpha of 5%.ResultsRANKL, RANKL:OPG ratio, COX-2, IL-6, DHCR7, CYP2R1 and AMDHD1 were differentially expressed during simulated orthodontic compressive strain (p < 0.05). The RANKL:OPG ratio was downregulated by all concentrations (10 ng/ml, 40 ng/ml and 60 ng/ml) of 25(OH)D3 (mean = 0.96 ± 0.68, mean = 1.61 ± 0.66 and mean = 1.86 ± 0.78, respectively) in comparison to the control (mean 2.58 ± 1.16) (p < 0.05). CYP2R1 gene expression was statistically modulated by the different 25(OH)D3 concentrations applied (p = 0.008). Samples from individuals carrying the GG genotype in rs739837 presented lower VDR mRNA expression and samples from individuals carrying the CC genotype in rs7975232 presented higher VDR mRNA expression (p < 0.05).ConclusionsSimulated orthodontic compressive strain and physiological concentrations of 25(OH)D3 seem to regulate the expression of orthodontic tooth movement and vitamin-D-related genes in periodontal ligament fibroblasts in the context of orthodontic compressive strain. Our study also suggests that single nucleotide polymorphisms in the VDR gene regulate VDR expression in periodontal ligament fibroblasts in the context of orthodontic compressive strain.

The VDR gene confers a genetic predisposition to Graves' disease and Graves' ophthalmopathy in the Southwest Chinese Han population.

Graves' disease (GD) is a common autoimmune disease manifesting with diffuse symmetric thyroid gland enlargement, pretibial myxedema, and Graves' ophthalmopathy (GO). Recently, the vitamin D receptor (VDR) gene has been linked to various autoimmune diseases. This study aimed to investigate the association of VDR gene polymorphisms with susceptibility to GD and GO in the Southwest Chinese Han population. A two-stage association study was performed in 1,209 controls and 650 GD patients by PCR-RFLP assay. Real-time PCR and ELISA were carried out to quantify gene expression and cytokine production. The first-stage study showed that the frequency of VDR/Apa I AA genotype was significantly increased in GD (Pc=1.67×10-2, OR=1.98). The second-stage and combined studies confirmed the association of VDR/Apa I with GD (AA genotype: Pc=3.45×10-4, OR=1.87; A allele: Pc=2.62×10-2, OR=1.20). The stratification analysis showed that GO patients had a higher frequency of the VDR/Apa I AA genotype (Pc=8.69×10-5, OR=2.84). Functional experiments showed a decreased VDR expression and TGF-β1 production as well as an increased IL-17 production in VDR/Apa I AA genotype carriers. The VDR/Apa I polymorphism is significantly associated with GD and GO, and it may be involved in the development of GD and GO by influencing VDR mRNA expression levels and the secretion levels of cytokines.

Vitamin D receptor and 1α-hydroxylase are highly expressed in lungs of mice infected with H9N2 avian influenza viruses

The H9N2 avian influenza viruses infect poultry worldwide, and can potentially cause a human pandemic without adaptation. Vitamin D3 (D3) is increasingly being recognized for its extra-skeletal roles, such as the inflammatory and immune responses to infection. The aim of this study was to analyze the changes in vitamin D metabolizing enzymes and vitamin D receptor (VDR) in the lung tissues of mice infected with H9N2. The mice were intranasally inoculated with the appropriate dose of the virus, and various clinical indices were measured on days 3, 7, 14 and 21 post-infection. H9N2 infection significantly increased the expression levels of 1α-hydroxylase mRNA and protein, which is the activating enzyme of 25-hydroxyvitamin D (25(OH)D3), but had no significant effect on the 25(OH)D3 inactivating enzyme 24-hydroxylase, indicating that inactive D3 might be converted to its active form in the H9N2-infected lungs. Furthermore, a significant increase was also observed in the VDR mRNA and protein levels, suggesting enhanced responsiveness of the lung tissues to 1, 25(OH)2D3 post H9N2 infection. In addition, daily 25(OH)D3 injection from day 2–14 post-infection did not affect the clinical signs, virus replication and cytokine (IL-1β and TNF-α) production in the lungs of the infected mice. Given that the biological effects of D3 rely on its activation, and the binding of 1, 25(OH)2D3 to VDR in specific tissues, our findings provide novel insights into the possible role of vitamin D in the development and progression of influenza.

Redefining the targets of the vitamin D receptor (VDR) utilizing a novel VDR reporter mouse.

Abstract The vitamin D receptor (VDR) is a ligand-activated nuclear protein that regulates gene transcription. Vitamin D target cells are identified by the expression of VDR. Kidney and colon epithelial cells constitutively express high levels of the VDR. Immune cells also express the VDR; however, at significantly lower levels. Western blots show very low expression of the VDR in spleen, and lymph nodes. Gene expression studies in macrophage, B cells and T cells showed that activation for 24–48 hours increased the VDR mRNA levels. We have developed transgenic mice that express Cre recombinase within the VDR locus (VDRcre) and crossed these with a reporter line (Tdtomatofl/fl). Cre expression in the VDR locus results in a VDR +/− mice. The frequencies of immune cells were similar between VDR−/− and VDR +/− mice. The VDRcre expressing offspring had pink skin. Kidneys, small intestine and colon were also distinctly pink suggesting high expression of the Tdtomato-VDR reporter. Splenic T cells, activated with CD3e antibody showed upregulation of VDR mRNA and protein. Regardless of activation status, naive, effector or memory T cells had similar and high levels of the VDR reporter. VDR expression was detected at various stages of T cell thymic development with a 80% in the CD4-CD8- cells and 90% of CD4+CD8+ cells, 84% CD4+ and 88% CD8+ thymocytes. High expression of the VDR reporter in multiple T cell types and precursors suggests that the VDR is expressed throughout the life of a T cell. Future work will identify immune cells negative for the VDR reporter and the kinetics of VDR expression following activation in various cell types.

MicroRNA-122 promotes apoptosis of keratinocytes in oral lichen planus through suppressing VDR expression.

MicroRNA‐122 (miR‐122) is known to be up‐regulated by inflammation to exert a variety of biological functions in hepatocellular carcinoma (HCC)‐derived human cell lines. Vitamin D receptor (VDR) is reported to regulate excessive oral keratinocytes apoptosis which compromises oral epithelial barrier in oral lichen planus (OLP). Although many studies have suggested that miR‐122 is capable of regulating cell apoptosis, its effects on the development of OLP and VDR expression are still unclear. Herein, we demonstrate that miR‐122 expression is increased in the epithelial layer of OLP. Mechanically, transcription factor nuclear factor‐κB (NF‐κB) selectively binds with κB element in the promoter of miR‐122 to accelerate gene transcription. The up‐regulation of miR‐122 induces cell apoptosis in human oral keratinocytes (HOKs) by targeting VDR mRNA. In VDR knockout oral keratinocytes, miR‐122 fails to improve caspase 3 activity and cleaved caspase 3 and poly(ADP‐ribose) polymerase (PARP) levels. Moreover, VDR overexpression is able to reverse lipopolysaccharide (LPS)‐ or activated CD4+ T cell–induced miR‐122 up‐regulation and ameliorate miR‐122‐stimulated caspase 3 activity. Collectively, our results suggest that miR‐122 promotes oral keratinocytes apoptosis in OLP through decreasing VDR expression.

Down regulation of VDR gene expression in metabolic syndrome and atherosclerosis' patients: Cause or consequence?

Metabolic syndrome (MS) features a set of clinical manifestations with an increased risk of atherosclerosis development. Vitamin D3 (VD3) pathway influences upon clinical features in MS as well in the formation of atherosclerotic plaque. VD3 acts through the vitamin D receptor (VDR), regulating the transcription of several genes involved in the immune response, growth and homeostasis. To evaluate whether VDR mRNA levels vary in MS patients according clinical features and atherosclerosis severity. We included eighty individuals distributed into four groups: 1 group with MS (n=20), 2 groups with atherosclerosis based on the report from the angiography (severe lesions - SL and primary lesions - PL) and 1 group enclosing healthy individuals (HC). The expression assays of VDR gene was performed using Real Time qPCR, with specific Taqman probes. We applied the Shapiro-Wilk, Chi-Squared and Student's T-tests for statistical analyses considering as statistically significant p<0.05. Patients with MS as well with coronary stenosis present a down-regulation of VDR gene expression (-9.01 FC, p=2.497×10-13; -13.62 FC, p=7.489×10-13, respectively) when compared to control group. We also evaluated the VDR gene expression according to atherosclerosis severity, SL and PL patients present a downregulation of -31.51 FC and -8.48 FC, respectively, when compared with healthy controls group (p=1.369×10-11; p=1.647×10-11). When compared different degrees of atherosclerosis severity (SL versus PL) SL present a downregulation of -3.71 FC, when compared to PL group (p=0.006). VDR is downregulated in patients with MS and according atherosclerosis severity. The differential expression of this gene is related to this hormone functions being an ex-vivo gene target for assessment in MS and atherosclerosis.

Association of vitamin D receptor and CYP2R1 mRNA expression with pulmonary tuberculosis

IntroductionThe vitamin D metabolic pathway has been shown to play a pivotal role in the pathogenesis of pulmonary tuberculosis (PTB), and vitamin D receptor (VDR) and CYP2R1 gene variation are known to affect vitamin D status. Hence, this study aimed to evaluate VDR and CYP2R1 mRNA expression in peripheral blood mononuclear cells (PBMCs) in PTB patients.Material and methodsWe measured VDR and CYP2R1 mRNA levels in 75 PTB patients and 63 healthy controls by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The associations of VDR and CYP2R1 mRNA levels with clinical characteristics and laboratory indexes of PTB patients were also examined in this study.ResultsCompared to healthy controls, the VDR mRNA level was significantly higher, and the CYP2R1 mRNA level was significantly lower in PBMCs from PTB patients (p = 0.047, p = 0.008, respectively). The CYP2R1 mRNA level in PTB patients with drug-resistant, unilateral tuberculosis foci was significantly higher than that in PTB patients without these clinical characteristics (p = 0.005, p = 0.048, respectively). In addition, our results demonstrated that the VDR mRNA expression level was positively correlated with erythrocyte sedimentation rate (ESR) (p = 0.045), while the CYP2R1 mRNA level was negatively correlated with ESR in PTB patients (p = 0.020).ConclusionsAltered VDR and CYP2R1 mRNA expression levels among PTB patients suggest their involvement in this disease.

Vitamin D receptor knockdown attenuates the antiproliferative, pro‑apoptotic and anti‑invasive effect of vitamin D by activating the Wnt/β‑catenin signaling pathway in papillary thyroid cancer.

Vitamin D and the vitamin D receptor (VDR) complex have been reported to inhibit the growth of several types of tumor; however, their function in papillary thyroid cancer (PCT) remains unknown. In addition, the Wnt/β-catenin signaling pathway was discovered to serve a critical role in the pathology of PCT. Therefore, the present study aimed to determine the role of the VDR and its association with Wnt/β-catenin signaling in vitamin D-treated PTC cells. VDR expression was detected in human PTC cells (including MDA-T120, MDA-T85, SNU-790 and IHH4 cells) and thyroid follicular cells (Nthy-ori 3–1 cells). SNU-790 and IHH4 cells were infected with KD-VDR or negative control (KD-NC) lentiviruses, treated with 1,25(OH)2D3 (the active form of vitamin D), and subsequently referred to as the KD-VDR&vitD and KD-NC&vitD groups, respectively. Additionally, PTC cells infected with KD-NC and not treated with 1,25(OH)2D3 were used as the normal control and referred to as the KD-NC group. VDR mRNA and protein expression levels were increased in MDA-T120, SNU-790 and MDA-T85 cells compared to Nthy-ori 3-1 cells, whereas in IHH4 cells, VDR mRNA and protein expression levels were similar to Nthy-ori 3-1 cells. In SNU-790 and IHH4 cells, cell proliferation and invasion were decreased in the KD-NC&vitD group compared with the KD-NC group, but increased in the KD-VDR&vitD group compared with the KD-NC&vitD group. Cell apoptosis was increased in the KD-NC&vitD group compared with the KD-NC group, and decreased in the KD-VDR&vitD group compared with the KD-NC&vitD group. Furthermore, the expression levels of Wnt family member 3 and catenin β1 were decreased in the KD-NC&vitD group compared with the KD-NC group, but increased in the KD-VDR&vitD group compared with the KD-NC&vitD group. In conclusion, the present study revealed that VDR-KD attenuated the antiproliferative, pro-apoptotic and anti-invasive effects of vitamin D in PTC by activating the Wnt/β-catenin signaling pathway.

Placenta expression of vitamin D and related genes in pregnant women with gestational diabetes mellitus

Objectives and MethodsA total of 41 GDM and 40 normal glucose tolerance subjects were recruited. Through detecting the level of Serum vitamin D with electrochemical luminescence and vitamin D receptor (VDR) with Enzyme-linked immunosorbent assay (ELISA) in maternal and cord blood, the expression leves of CYP24A1, CYP27B1, VDR protein and mRNA in placenta and umbilical cord with western blotting and RT-PCR, and the DNA methylation levels of CYP24A1 and CYP27B1 gene in placenta with methylation-specific PCR (MSP) and direct bisulfite sequencing (BSP) analysis to explore the potential role of the vitamin D and its related genes in gestational diabetes mellitus (GDM). ResultsSerum vitamin D concentrations were significantly higher in normal pregnant than women with GDM in maternal blood (P < 0.01) and cord blood (P = 0.014). Compared to the control group, the expression levels of CYP24A1 protein (P < 0.01) and mRNA (P = 0.021) and VDR protein (P = 0.026) and mRNA (P = 0.023) in the GDM group were significantly higher in placenta and umbilical cord tissues (P = 0.015, P < 0.01, P = 0.028, P < 0.01, respectively), while that of CYP27B1 protein (P < 0.01) and mRNA (P = 0.042) was significantly lower (P = 0.022, P = 0.032, respectively). Moreover, partial DNA methylation of CYP24A1 and CYP27B1 genes was observed in both GDM and control groups. ConclusionsVitamin D deficiency participates in the pathogenesis of GDM, and changes in the expression of genes related to the vitamin D metabolic pathway are closely related to vitamin D levels in the pregnancy and fetus. However, DNA methylation of CYP24A1 and CYP27B1 might not be involved in the pathogenesis of GDM.

Differential distribution in vitamin D receptor gene variants and expression profile in Northeast Brazil influences upon active pulmonary tuberculosis.

Tuberculosis is an infectious disease with variable outcomes. This variability is due to host immune capacity in containing the infection process initiated by the Mycobacterium tuberculosis (MTB). Vitamin D is able to modulate a very specific immune response against MTB infection, and its action relies on vitamin D receptor (VDR) binding. Altered VDR forms may compromise vitamin D pathway and proper immune response after MTB infection. Herein we assessed the relationship of five potentially functional polymorphisms from VDR: rs2228570 FokI, rs11568820 Cdx-2, rs2248098, rs1540339 and rs4760648, with tuberculosis susceptibility. The SNP rs4760648 T/T was associated with differential susceptibility to tuberculosis (OR = 2.50, 95%CI = 1.20-5.36, p = 0.01). The SNP rs1540339 presented association to both T allele (OR = 0.55, 95%CI = 0.35-0.88, p = 0.01) and the T/T genotype (OR = 0.404, 95%CI = 0.20 - 0.78, p = 0.005). The FokI T allele was identified as associated to diminished susceptibility (OR = 0.67, 95% CI = 0.45-0.99, p = 0.04) to active TB, as well as T/T genotype (OR = 0.15, 95%CI = 0.04-0.45, p = 9.58 × 10-5). We also performed the expression analyses and observed a down-regulation of VDR in patients (-10.717 FC, p = 8.42e-12), and according to the presence of associated FokI SNP, we observed that the C/T and T/T genotypes presence increases VDR expression (+ 1.25 and + 2.35 FC, p = 0.425 and p = 0.506, respectively). This study shows that vitamin D receptor variants can influence upon pulmonary tuberculosis susceptibility and VDR mRNA levels are decreased in those patients.

Overexpression of the vitamin D receptor (VDR) induces skeletal muscle hypertrophy

ObjectiveThe Vitamin D receptor (VDR) has been positively associated with skeletal muscle mass, function and regeneration. Mechanistic studies have focused on the loss of the receptor, with in vivo whole-body knockout models demonstrating reduced myofibre size and function and impaired muscle development. To understand the mechanistic role upregulation of the VDR elicits in muscle mass/health, we studied the impact of VDR over-expression (OE) in vivo before exploring the importance of VDR expression upon muscle hypertrophy in humans. MethodsWistar rats underwent in vivo electrotransfer (IVE) to overexpress the VDR in the Tibialis anterior (TA) muscle for 10 days, before comprehensive physiological and metabolic profiling to characterise the influence of VDR-OE on muscle protein synthesis (MPS), anabolic signalling and satellite cell activity. Stable isotope tracer (D2O) techniques were used to assess sub-fraction protein synthesis, alongside RNA-Seq analysis. Finally, human participants underwent 20 wks of resistance exercise training, with body composition and transcriptomic analysis. ResultsMuscle VDR-OE yielded total protein and RNA accretion, manifesting in increased myofibre area, i.e., hypertrophy. The observed increases in MPS were associated with enhanced anabolic signalling, reflecting translational efficiency (e.g., mammalian target of rapamycin (mTOR-signalling), with no effects upon protein breakdown markers being observed. Additionally, RNA-Seq illustrated marked extracellular matrix (ECM) remodelling, while satellite cell content, markers of proliferation and associated cell-cycled related gene-sets were upregulated. Finally, induction of VDR mRNA correlated with muscle hypertrophy in humans following long-term resistance exercise type training. ConclusionVDR-OE stimulates muscle hypertrophy ostensibly via heightened protein synthesis, translational efficiency, ribosomal expansion and upregulation of ECM remodelling-related gene-sets. Furthermore, VDR expression is a robust marker of the hypertrophic response to resistance exercise in humans. The VDR is a viable target of muscle maintenance through testable Vitamin D molecules, as active molecules and analogues.

'Psoriasis1' reduces T‑lymphocyte‑mediated inflammation in patients with psoriasis by inhibiting vitaminD receptor‑mediated STAT4 inactivation.

Psoriasis is an immune-mediated dermatosis characterized by T-lymphocyte-mediated epidermal hyperplasia, for which there are currently no effective clinical treatments. 'Psoriasis 1' is a Chinese herbal medicine formulation that has been recently used extensively in China for treating patients with psoriasis. However, the molecular mechanism of action of this potent formulation has not yet been fully elucidated. In the present study, the effects of 'Psoriasis 1' on T ymphocytes in patients with psoriasis were investigated and the underlying molecular mechanism was discussed. Blood samples were collected from 40 patients with psoriasis. ELISA was employed to assess the levels of tumour necrosis factor-α, interferon-γ, interleukin (IL)-2, IL-6, transforming growth factor-β, IL-4, IL-12, IL-23 and vitamin D (VD). Western blot and quantitative PCR analyses were used to investigate the expression levels of VD receptor (VDR) and signal transducer and activator of transcription (STAT)4 in T lymphocytes. 'Psoriasis 1' was observed to significantly increase CD4+ T cells. It also notably upregulated the mRNA and protein expression of VDR, and downregulated the mRNA and protein expression of STAT4. Moreover, the suppression of VDR was found to aggravate the inflammatory response, which was reversed by 'Psoriasis 1.' Thus, this formulation reportedly decreased the inflammation mediated by T lymphocytes in patients with psoriasis through inhibiting VDR-mediated STAT4 inactivation.

Relationship between hepatic and systemic angiopoietin-like 3, hepatic Vitamin D receptor expression and NAFLD in obesity.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide and an independent risk factor for cardiovascular mortality. Angiopoietin-like proteins (ANGPTLs) are targets for vitamin D receptor (VDR)-mediated gene transcription and this axis may promote NAFLD. ANGPTL3 is a hepatokine which inhibits lipoprotein lipase and its experimentally induced inactivation reduces hepatosteatosis. Little is known on ANGPTL3 in human NAFLD and no data exist on its relationship with hepatic VDR/VD-related genes. The aim of this research was to investigate hepatic ANGPTLs and VDR/VD-related gene expression in human obesity in relation to NAFLD. We conducted a cross-sectional investigation on forty obese subjects with/without NAFLD. We evaluated hepatic ANGPTL3, ANGPTL4, ANGPTL8, LPL, VDR, CYP27A1 and CYP2R1 mRNA expression in liver biopsies by RT-PCR; VDR expression was further investigated by immunohistochemistry; circulating ANGPTL3 was measured by Milliplex assay. Compared to non-NAFLD, NAFLD individuals had significantly higher hepatic VDR, ANGPTL3 and LPL expression. ANGPTL3 correlated with steatosis grade, LPL, VDR, CYP27A1 and CYP2R1 expression. Plasma ANGPTL3 concentrations were positively associated with clinical/histological markers of NAFLD/NASH and with hepatic ANGPTL3 expression. Greater hepatic VDR expression was the main determinant of hepatic ANGPTL3 after adjusting for multiple confounders. Hepatic ANGPTL3 expression correlates with greater VDR expression, presence and severity of NAFLD and translates in increased circulating ANGPTL3, likely as a result of its modulation by up-regulated hepatic VDR in NAFLD. This study provides novel insights to potential mechanisms underlying ANGPTLs-mediated ectopic fat accumulation and NAFLD development in obesity.

SAT-LB36 Distinct Vitamin D Receptor DNA Methylation Profiles Are Associated With the Outcome of Pediatric Patients With Adrenocortical Tumors

Pediatric adrenocortical tumors (pACT) are rare, display complex genomic background and lack robust prognostic markers. Very recently, distinct genomic methylation profiles of pACT were associated with prognosis. The vitamin D receptor (VDR) was shown to be underexpressed in ACT, especially in carcinomas (ACC). In adult ACC, VDR inactivation by methylation was demonstrated. On the other hand, VDR activation was shown to inhibit ACC proliferation in vitro and in vivo. Aim: To evaluate VDR DNA methylation profile and its clinical and prognostic significance in pediatric ACT. Methods: Genomic DNA methylation from 57 pACTs [40 girls; median age: 2.1 (0.2-16.4) years] was assessed using Infininium Methylation EPIC BeadChip Array. Unsupervised hierarchical clustering analysis (Ward method, R Stats Package) was performed considering the M-values of the 49 probes targeting the whole extension of VDR gene contained in the array. Clinical, histopathological and molecular features, as well as pACT VDR mRNA levels (qPCR) and nuclear immunoreactivity (IHC) were used for association analysis. Results: Hierarchical clustering identified three clusters of pACT. Methylated VDR-targeted probes (M-values different from 0; n=37) composed the VDR methylation profile, which differed significantly between the clusters [M-values: C1=1.77 (1.1-1.9) (low), C2=2.15 (1.7-2.7) (intermediate), and C3=2.65 (2.2-3.1) (high); p<0.0001]. The C1 cluster comprised a set of patients with favorable outcome (n=18), who were younger (p=0.035), did not present metastasis at diagnosis (IPACTR stage IV) or after surgery, nor were diagnosed with carcinomas (Wieneke criteria >=4), were not carriers of somatic Beta-catenin activating mutations, or died. Although cluster C2 patients (n=21) presented intermediary disease features, only 2 patients died and the overall outcome was positive. Instead, the C3 cluster concentrated patients (n=18) with non-localized/metastatic disease (IPACTR stages I/II vs. III/IV; p=0.004), post-surgical metastasis/recurrence (p=0.009), and patients who needed adjuvant chemotherapy (p=0.005). Moreover, C3 patients had lower overall and disease-free survival rates (log-rank: p=0.001 and p=0.014, respectively). VDR methylation was not associated with sex, clinical presentation, P53 mutations, nor with tumor VDR mRNA expression or nuclear immunoreactivity. Conclusions: Three VDR methylation profiles were associated with distinct pACT clinical features and outcome. High VDR methylation was associated with worst outcome. Fully functioning VDR may play a beneficial role against pediatric adrenocortical tumorigenesis. This finding highlights the potential of targeting VDR as an adjuvant therapeutic target.

Vitamin D receptor polymorphisms are associated with severity of wheezing illnesses and asthma exacerbations in children

Single nucleotide polymorphisms (SNPs) of the vitamin D receptor (VDR) gene have shown linkage and association with asthma development in multiple cohort studies. However, the majority of investigations have focused on asthma phenotypes in cohorts with stable disease. We investigated the relationship between VDR SNPs and the frequency and severity of acute episodes of wheeze/asthma in a cohort of Australian children, as the ability to identify children at risk of more severe exacerbations could lead to personalized and improved genotype-specific treatment pathways. We successfully genotyped five SNPs of the VDR gene (rs2525046, rs9729, rs1544410 (BsmI), rs22239179, and rs2228570 (FokI)) in 657 children presenting to a tertiary children’s hospital with acute asthma, bronchiolitis, or a wheezing illness. The relationships between VDR SNPs and exacerbation severity scores, β2-agonist use, and frequency of respiratory exacerbations were analysed using multiple regression. The rs2525046 (FokI) CT genotype was associated with higher VDR mRNA intensity levels (p = 0.007) compared to the CC genotype. A trend towards significance (p=0.056) was identified between the rs2525046 TT genotype and higher VDR mRNA intensity levels compared to the CC genotype. Children with rs2228570 AA genotype had higher exacerbation severity scores (p=0.001) and poorer β2-agonist treatment response (doses at 6 h: p = 0.009 and 12 h: p=0.033) compared to those with the GG genotype. Children with rs1544410 (BsmI) TT genotype had lower exacerbation severity scores (p = 0.005) compared to those with the CC genotype. Children with rs2228570 GA genotype presented to and/or were admitted to hospital more times since birth with respiratory (p = 0.011) and wheezing (p = 0.021) illnesses than children with the GG genotype. No associations were identified between rs9729, rs2525046 and r2239179 polymorphisms and acute wheezing/asthma variables. These findings suggest that genetic variants at the VDR locus may play a role in acute wheeze/asthma severity in children.

Vitamin D modulates innate immunity in pulmonary tuberculosis

Abstract Vitamin D and vitamin D receptor (VDR) may play role in altering host defence against Mtb via cathelicidin production and regulating nitric oxide (NO) production through inducible nitric oxide synthase (iNOS). 100 active pulmonary tuberculosis (PTB) patients, 75 household contacts and 70 healthy controls were recruited. Serum Vitamin D and NO levels were measured using CLIA and colorimetry based kit respectively. VDR and iNOS mRNA was studied in monocytes using qPCR. Serum VDR, cathelicidin and iNOS levels were measured using ELISA. Lower serum levels of vitamin D was found in PTB group compared to household contacts and healthy controls (P&amp;lt;0.001). VDR (both mRNA and protein) and iNOS mRNA levels were also lower in PTB group compared to household contacts and healthy controls (P&amp;lt;0.005)probably due to decreased levels of vitamin D. However, despite low serum vitamin D, serum cathelicidin levels were higher in PTB group compared to household contacts and healthy controls (p&amp;lt;0.01) which could be attributed to direct trigger via toll like receptors where Mtb is multiplying actively. Serum iNOS and NO levels were higher in PTB group compared to household contacts and healthy controls (p&amp;lt; 0.05), despite decreased iNOS expression which needs further evaluation. Increased VDR and iNOS expression in household contacts compared to both PTB and healthy controls could be due to optimum levels of vitamin D along with some exposure to Mtb as evident by increased levels of cathelicidin and NO compared to healthy controls. Adequate levels of vitamin D, VDR and iNOS that in turn promote production of antimicrobial cathelicidin and NO might have protective role against TB among household contacts and needs further investigation for its therapeutic potential.

Autophagy efficacy and vitamin D status: Population effects

Toll-like receptor (TLR) 2/1 signalling is linked to autophagy through transcriptional actions of the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-vitamin D receptor (VDR) complex. Population-specific effects have been reported for TLR2/1-VDR signalling. We hypothesized that population effects extend to autophagy and are influenced by vitamin D status. Serum 25(OH)D3 of healthy South Africans (Black individuals n = 10, White individuals n = 10) was quantified by LC-MS/MS. Primary monocytes-macrophages were supplemented in vitro with 1,25(OH)2D3 and stimulated with the lipoprotein Pam3CysSerLys4. TLR2, VDR, hCAP18, Beclin1, LC3-IIB, cytokines and CYP24A1 mRNA were quantified by flow cytometry and RT-qPCR, respectively. Black individuals showed significantly lower overall cumulative LC3-IIB (P < 0.010), but higher Beclin1, VDR, IL6 and TNFA (P < 0.050) than White individuals. 1,25(OH)2D3 enhanced autophagic flux in monocytes-macrophages from Black individuals upon TLR2/1 stimulation and strengthened autophagy in 25(OH)D3 deficient individuals (independent cohort, n = 20). These findings support population-directed vitamin D supplementation.

Increased brain vitamin D receptor expression and decreased expression of cathelicidin antimicrobial peptide in individuals who died by suicide

Vitamin D deficiency is associated with immune dysregulation, increased vulnerability to infections, depression, and suicidal behavior. One mediator of vitamin D-dependent immune regulation and antimicrobial defense is the cathelicidin antimicrobial peptide (LL-37), encoded by the cathelicidin-related antimicrobial peptide (CRAMP) gene. We compared the mRNA expression of the CRAMP gene, the vitamin D receptor (VDR) gene, as well as the CYP27B1 and CYP24A1 genes (involved in vitamin D metabolism) in the dorsolateral prefrontal cortex (dlPFC) and anterior cingulate cortex (ACC) between depressed individuals who died by suicide (n = 15) and matched (age, gender, and post-mortem interval) non-psychiatric controls (n = 15). Gene expression was measured through qRT-PCR with TaqMan® primers and probes, with GAPDH and β-actin genes as endogenous controls. Statistical analyses included t-tests and Pearson correlations. CRAMP mRNA expression was downregulated and VDR mRNA expression was upregulated in both dlPFC and ACC in suicides relative to controls, with no significant differences in expression of CYP24A1 and CYP27B1. To our knowledge, this is the first study on brain cathelicidin expression in the human brain in relationship to suicide. Increased VDR and decreased CRAMP expression are consistent with previously reported associations between vitamin D deficiency, immune dysregulation, and suicidal behavior, and should lead to future studies uncovering novel interactive targets for suicide prevention.

1,25-hydroxyvitamin D3 decreases endoplasmic reticulum stress-induced inflammatory response in mammary epithelial cells.

Recent studies indicated that intramammary administration of active vitamin D3 hormone (1,25D3) inhibits the inflammatory process associated with mastitis. We hypothesized that attenuation of endoplasmic reticulum (ER) stress by 1,25D3 in mammary epithelial cells (MECs) is an important cellular mechanism contributing to this beneficial effect of intramammary treatment with 1,25D3. To test this hypothesis, the effect of 1,25D3 was studied on induction of ER stress in a transformed human MEC line, MCF-7 cells. Treatment with two different ER stress inducers, thapsigargin (TG) and tunicamycin (TM), caused a dose-dependent induction of ER stress as evident from up-regulation of protein kinase RNA-like ER kinase (PERK), heat shock protein family A (Hsp70) member 5 (HSPA5), activating transcription factor (ATF4), ATF6, DNA damage inducible transcript 3 (DDIT3) and spliced X-box binding protein 1 (sXBP1) and impaired cell viability and decreased expression of vitamin D receptor (VDR) in MCF-7 cells (P < 0.05). Treatment with 1,25D3 (100 nM) inhibited TG (10 nM)- and TM (1 μg/mL)-induced mRNA and/or protein levels of ATF4, ATF6, DDIT3 and HSPA5 in MCF-7 cells (P < 0.05). In addition, 1,25D3 (100 nM) antagonized the effect of TG (10 nM) and TM (1 μg/mL) on mRNA and protein levels of VDR and mRNA levels of genes involved in production and degradation of 1,25D3 in MCF-7 cells (P < 0.05). Moreover, 1,25D3 (100 nM) inhibited nuclear factor-κB (NF-κB) activation in response to TM (10 nM) and TG (1 μg/mL) in MCF-7 cells. In conclusion, the present findings show that 1,25D3 is effective in attenuating ER stress and the NF-κB-driven inflammatory response in MCF-7 cells. This indicates that attenuation of ER stress by 1,25D3 in MECs may contribute to the recently observed inhibitory effect of intramammary treatment of dairy cows with 1,25D3 on the inflammatory process associated with mastitis.

Vitamin D receptor and monocyte chemoattractant protein-1 expressions in peripheral blood mononuclear cells in patients with systemic lupus erythemaotsus

To detect the expressions of vitamin D receptor (VDR) in peripheral blood monocytes (PBMCs) and its association with monocyte chemoattractant protein-1 (MCP-1) in patients with systemic lupus erythemaotsus (SLE). We examined the expressions of VDR and MCP-1 mRNAs in the PBMCs in 60 SLE patients and 28 healthy individuals using real- time quantitative PCR. We also detected the expression of VDR protein in the PBMCs using Western blotting and peripheral blood MCP-1 level using ELISA for these participants. The correlation of VDR and MCP-1 expressions with the disease activity index of SLE (SLEDAI) of the patients were analyzed. The expressions of VDR mRNA and protein in the PBMCs were significantly lower in patients with SLE than in the healthy individuals (P < 0.01), and that in the patients with active disease was lower than in remission (P < 0.05). The MCP-1 mRNA expression in the PBMCs and its serum levels were significantly increased in SLE patients as compared with the healthy individuals (P < 0.01), and the increase was significantly more obvious in the patients with active disease than in those in remission (P < 0.01). Pearson correlation analysis showed that VDR mRNA in the PBMCs was negatively correlated with SLEDAI (r=-0.417, P=0.001); negative correlations were also found between VDR mRNA and MCP-1 mRNA(r=-0.554, P=0.000) and between VDR protein expression and serum MCP-1 level (r=-0.400, P=0.028). The down-regulation of VDR expression in the PBMC is negatively correlated with the disease activity of SLE. VDR may play an important role in the pathogenesis of SLE by affecting the expression of MCP-1.