Vitamin D Receptor Protein Levels Research Articles

Gene expression profiling of vitamin D metabolism enzymes in leukemia and lymphoma patients: molecular aspect interplay of VDR, CYP2R1, and CYP24A1.

Vitamin D deficiency is prevalent among the Indonesian population, particularly in individuals diagnosed with leukemia-lymphoma. The regulation of vitamin D metabolism is influenced by the expression of several enzymes, such as CYP2R1, CYP24A1, and the vitamin D receptor (VDR). This study aimed to scrutinize the gene expression profiles in both mRNA and protein levels of VDR, CYP2R1, and CYP24A1 in leukemia and lymphoma patients. The research was a cross-sectional study conducted at Cipto Mangunkusumo Hospital (RSCM) in Jakarta, Indonesia. The study included a total of 45 patients aged over 18years old who have received a diagnosis of lymphoma or leukemia. Vitamin D status was measured by examining serum 25 (OH) D levels. The analysis of VDR, CYP2R1, and CYP24A1 mRNA expression utilized the qRT-PCR method, while protein levels were measured through the ELISA method. The study revealed a noteworthy difference in VDR protein levels between men and women. The highest mean CYP24A1 protein levels were observed in the age group > 60years. This study found a significant, moderately positive correlation between VDR protein levels and CYP24A1 protein levels in the male and vitamin D sufficiency groups. In addition, a significant positive correlation was found between VDR mRNA levels and CYP2R1 mRNA levels, VDR mRNA levels and CYP2R1 mRNA levels, and CYP2R1 mRNA levels and CYP24A1 mRNA levels. However, the expression of these genes does not correlate with the protein levels of its mRNA translation products in blood circulation.

The Vitamin D Receptor as a Prognostic Marker in Breast Cancer-A Cohort Study.

Previous research has indicated an association between the presence of the vitamin D receptor (VDR) in breast cancer tissue and a favorable prognosis. This study aimed to further evaluate the prognostic potential of VDR located in the nuclear membrane or nucleus (liganded). The VDR protein levels were analyzed using immunohistochemistry in tumor samples from 878 breast cancer patients from Lund, Sweden, included in the Breast Cancer and Blood Study (BCBlood) from October 2002 to June 2012. The follow-up for breast cancer events and overall survival was recorded until 30 June 2019. Univariable and multivariable survival analyses were conducted, both with complete case data and with missing data imputed using multiple imputation by chained equations (MICE). Tumor-specific positive nuclear membrane VDR(num) staining was associated with favorable tumor characteristics and a longer breast cancer free interval (BCFI; HR: 0.64; 95% CI: 0.44-0.95) and overall survival (OS; HR: 0.52; 95% CI: 0.34-0.78). Further analyses indicated that VDRnum status also was predictive of overall survival when investigated in relation to ER status. There were significant interactions between VDR and invasive tumor size (Pinteraction = 0.047), as well as mode of detection (Pinteraction = 0.049). VDRnum was associated with a longer BCFI in patients with larger tumors (HR: 0.36; 95% CI: 0.14-0.93) or clinically detected tumors (HR: 0.28; 95% CI: 0.09-0.83), while no association was found for smaller tumors and screening-detected tumors. Further studies are suggested to confirm our results and to evaluate whether VDR should and could be used as a prognostic and targetable marker in breast cancer diagnostics.

The Influence of Single Nucleotide Polymorphisms on Vitamin D Receptor Protein Levels and Function in Chronic Liver Disease.

Single nucleotide polymorphisms (SNPs) in the vitamin D receptor (VDR) gene have been associated with chronic liver disease. We investigated the role of VDR SNPs on VDR protein levels and function in patients with chronic liver disease. VDR expression levels were determined in peripheral T lymphocytes (CD3+VDR+), monocytes (CD14+VDR+), and plasma from patients (n = 66) and healthy controls (n = 38). Genotyping of SNPs and the determination of expression of VDR/vitamin D-related genes were performed by using qPCR. The effect of FokI SNP on vitamin D-binding to VDR was investigated by molecular dynamics simulations. CD14+VDR+ cells were correlated with the MELD score. The ApaI SNP was associated with decreased CD3+VDR+ levels in cirrhotic patients and with higher liver stiffness in HCV patients. The BsmI and TaqI SNPs were associated with increased VDR plasma concentrations in cirrhotic patients and decreased CD14+VDR+ levels in HCV patients. The FokI SNP was associated with increased CD3+VDR+ levels in cirrhotic patients and controls. VDR polymorphisms were significantly related to the expression of genes critical for normal hepatocyte function and immune homeostasis. VDR expression levels were related to the clinical severity of liver disease. VDR SNPs may be related to the progression of chronic liver disease by affecting VDR expression levels.

#5645 SHORT-CHAIN FATTY ACIDS, PROPIONATE AND BUTYRATE, RESEMBLE THE BENEFICIAL EFFECTS OF VITAMIN D DURING ACUTE KIDNEY DAMAGE

Abstract Background and Aims The vitamin D receptor (VDR) is a nuclear receptor that acts as a ligand-induced transcription factor regulating the renal expression of numerous genes with anti-inflammatory and anti-fibrotic effects, among others. For that, VDR requires the binding of its ligand, 1,25 (OH)2D3, to further heterodimerize with its co-activator, RXR, and translocate into the nucleus. Vitamin D deficiency in patients with renal disease leads to a decrease in VDR-mediated signaling and, consequently, of its beneficial functions. Additionally, these patients have intestinal dysbiosis, which leads to alterations in the production of microbial metabolites. Short-chain fatty acids (SCFAs) are metabolites with a clear anti-inflammatory and immunoregulatory role. Until now, some studies have studied their role as regulators of the inflammation and oxidative stress during the renal damage.1,2 However, their role in the VDR-mediated signaling in the kidney and the potential consequences to prevent the progression of the disease have not yet been explored in detail. Method The tubular epithelial cell line, HK2, was used to analyze the effect of the SCFAs, Propionate (Prop,1-15mM) and Butyrate (But, 0.5-3 mM), in the VDR expression and their target-genes. In vitro treatment with histone deacetylases (HDACs) inhibitors and specific HDAC1 and HDAC3 siRNAs were used to determine the role of both SCFAs as epigenetic remodelers. The binding of VDR to HDAC1/3 and RXR was determined by ChIP and co-immunoprecipitation assays. The in vivo effect of these SCFAs was evaluated in an acute kidney injury mice model induced by folic acid (250 mg/kg) and administration of Prop (200mg/kg) or But (500mg/kg) at different time points. The expression of VDR and its target genes, inflammatory cell infiltration, renal damage markers and renal function parameters were evaluated at shorter (24h) and longer times (40 days). Results Treatment of tubular cells with Prop and But induces, in a dose-dependent manner, the VDR gene transcription. This effect is similar to the one obtained by using specific inhibitors and siRNA treatments against HDAC1/3. We determine using ChIP assay, that HDAC1/3 are recruited to the promoter regions of the VDR gene, blocking its expression. In presence of Prop and But, these HDACs are displaced, increasing the acetylation levels and VDR transcription. Moreover, Prop and But prevent the degradation of VDR by the proteasome, increasing its stability and enhancing VDR protein levels. In the presence of both SCFAs, VDR dimerizes with RXR initiating its translocation to the nucleus and allowing the transcription of its dependent genes, such as Cyp24a1 and E-cadherin. Of note, VDR activation by Prop and But is additive to the effect achieved by the vitamin D alone. In vivo studies reported that administration of Prop or But prevents the loss of VDR expression 24h after induction of the damage, leading to its activation and to the expression of its target genes. Additionally, a decrease in the recruitment of neutrophils to the kidney was observed associated to a reduced expression of IL-6. These changes are accompanied by a decrease of the kidney damage markers (KIM-1 and NGAL) and a significant reduction of the creatinine and blood urea nitrogen serum levels. In a second model of AKI-to CKD transition, administration of both SCFAs shows a decrease in the inflammatory (Ccl20, Ccl2, Lif, Ltb, Csf2SF2, Il18, Ccl5, Tnf-α) and pro-fibrotic markers (Fsp1, α-Sma, Col1a1, Fn1) and a partial recovery of the glomerular filtration rate at long term. Conclusion Propionate and Butyrate, not only induce the VDR gene transcription in renal tubular cells but are also able to stabilize and activate the VDR protein. Accordingly, both metabolites are able to restore the loosed expression and activation of VDR due to induced renal damage, reduce the infiltration of immune cells, and partially recover the renal function. Thus, strategies aimed to increase the propionate and butyrate levels with postbiotics could be useful to ameliorate the AKI renal damage and CKD transition.

High Vitamin D Level in Female Mice Increases the Number of Live Fetuses.

Little is known about the impact of high-normal range of 25-hydroxyvitamin D [25(OH)D] on reproductive function. The aim of this study was to investigate the effect of different dose vitamin D supplementation in female mice on the pregnancy outcomes. Three groups of female mice were fed with fodder containing different dose of vitamin D at both pre-gestational and gestational stages. Serum 25(OH)D and calcium concentrations were monitored. The expression levels of vitamin D receptor (VDR) mRNA and protein in placenta were determined by real-time RT-PCR and western blot. Pregnancy outcomes were evaluated and compared among the three groups. Compared with the medium and low dose groups, serum 25(OH)D concentration was significantly increased and approximated to high-normal range in the high dose group (pre-gestational: 81.3±5.75 vs 52.8±6.24 and 25.0±3.99 ng/mL; gestational: 86.8±5.99 vs 52.6±9.29 and 27.9±4.96 ng/mL, respectively; all p<0.001). Interestingly, the average number of live fetuses per litter was much larger in the high dose group than in other two groups (19.8±5.31 vs 13.8±1.30 and 12.8±3.55 respectively, both p<0.05). However, no significant differences of the expression levels of VDR mRNA and protein in placenta were identified among the three groups. Supplementation of high dose vitamin D can enhance the female mice reproductive function. Further study is warranted to explore the mechanism by which high level of 25(OH)D in female mice increases the number of fetuses.

Clinical efficacy of moxibustion for ulcerative colitis and its influence on vitamin D receptor

ObjectiveTo observe the clinical efficacy of herbal cake-partitioned moxibustion for ulcerative colitis (UC) and elucidate its mechanism by targeting the vitamin D receptor (VDR) signaling pathway.MethodsA total of 63 patients with UC were randomly divided into an observation group (30 cases, treated with herbal cake-partitioned moxibustion) and a control group (33 cases, treated with sham herbal cake-partitioned moxibustion). Moxibustion treatment was performed at Qihai (CV6) and bilateral Tianshu (ST25) and Shangjuxu (ST37), 3 times per week for 12 weeks. The total effective rate, visual analog scale (VAS) score for abdominal bloating and pain, and hospital anxiety and depression scale (HADS) score were compared between the two groups. Enzyme-linked immunosorbent assay was used to detect the concentrations of serum C-reactive protein (CRP), 25-hydroxyvitamin D [25(OH)D], and interleukin-12 (IL-12)/interleukin-23 (IL-23) p40. Immunohistochemistry was used to observe the expression levels of VDR and regenerating gene IV (Reg IV) proteins in colonic mucosa. The expression levels of VDR, cytochrome p450 27B1 (CYP27B1), and Reg IV mRNAs were detected by real-time fluorescence quantitive polymerase chain reaction.ResultsAfter treatment, the total effective rate in the observation group was 86.7%, which was significantly higher than 51.5% in the control group (P<0.05). After treatment, the VAS scores for abdominal bloating and pain in the observation group were significantly decreased (P<0.01), as well as the HADS-depression subscale (HADS-D) and HADS-anxiety subscale (HADS) scores (P<0.05), while only the VAS score for abdominal pain in the control group was reduced (P<0.05), and the improvements of the scores in the observation group were more significant than those in the control group (P<0.05). After treatment, the serum CRP concentrations in both groups and the IL-12/IL-23 p40 concentration in the observation group were significantly decreased (P<0.05), and the concentrations in the observation group were lower than those in the control group (P<0.05). The expression levels of VDR protein and mRNA in the colon in both groups were all increased (P<0.01), and the expression levels of Reg IV protein and mRNA and CYP27B1 mRNA were all decreased in the two groups (P<0.05 or P<0.01); the improvements in the observation group were more notable than those in the control group (P<0.05 or P<0.01).ConclusionHerbal cake-partitioned moxibustion can effectively alleviate abdominal pain and diarrhea in patients with UC, improve depression and anxiety disorders, and regulate the expression of related proteins in the VDR signaling pathway. The mechanism may be related to inhibiting intestinal inflammation by reducing the release of the proinflammatory cytokine IL-12/IL-23 p40.

MiR-27a-3p and miR-30b-5p inhibited-vitamin D receptor involved in the progression of tuberculosis.

BackgroundMicroRNAs (miRNAs) play a vital role in tuberculosis (TB). Vitamin D receptor (VDR), an miRNA target gene, and its ligand, vitamin D3 (VitD3), have been reported to exert protective effects against TB. However, whether miRNAs can affect the progression of TB by targeting VDR has not been reported.Materials and methodsResearch subjects were selected according to defined inclusion criteria. A clinical database of 360 samples was established, including the subjects’ demographic information, miRNA expression profiles and cellular experimental results. Two candidate miRNAs, miR-27a-3p, and miR-30b-5p, were identified by a high-throughput sequencing screen and validated by qRT–PCR assays. Univariate and multivariate statistical analyses were performed. VDR and NF-kB p65 protein levels were detected by Western blot assays. Proinflammatory cytokine expression levels were detected by enzyme-linked immunosorbent assay (ELISA). Luciferase assays and fluorescence-activated cell sorting (FACS) were further applied to elucidate the detailed mechanisms.ResultsDifferential miRNA expression profiles were obtained, and miR-27a-3p and miR-30b-5p were highly expressed in patients with TB. These results showed that the two miRNAs were able to induce M1 macrophage differentiation and inhibit M2 macrophage differentiation. Further experiments showed that the two miRNAs decreased the VDR protein level and increased proinflammatory cytokine secretion by macrophages. Mechanistically, the miRNAs targeted the 3′ untranslated region (3′UTR) of the VDR mRNA and thereby downregulated VDR protein levels by post-transcriptional regulation. Then, due to the reduction in VDR protein levels, the NF-kB inflammatory cytokine signaling pathway was activated, thus promoting the progression of TB.ConclusionOur study not only identified differentially expressed miRNAs between the TB and control groups but also revealed that miR-27a-3p and miR-30b-5p regulate proinflammatory cytokine secretion and macrophage differentiation through VDR in macrophages. Thus, these two miRNAs influence the progression of TB.

The Role of Polylactic-Coglycolic Acid-Coated Electrospinning Nanoscaffold-Mediated Wnt/β-Catenin Signaling Pathway in Intervertebral Disc Degeneration and Its Relationship with Vitamin D Receptor

Using polyvinyl alcohol (PVA), polylactic coglycolic acid (PLGA), and transforming growth factor-β (TGF-β1) as raw materials, PLGA-coated electrospun nanoscaffold (ESNS) PVA-TGF-β1@PLGA was prepared by the coaxial ES method. Its characterization was analyzed by scanning electron microscopy (SEM), high-performance liquid chromatography (HPLC), and Fourier transform infrared spectroscopy (FTIR). Seventy-five healthy New Zealand rabbits were randomly rolled into the control group (no treatment, group C), model group (intervertebral disc degeneration (IDD) model, group M), and nanofiber scaffold group (implant PVA- TGF-β1@PLGA, group P). HE staining was adopted to visualize the histological morphology of the intervertebral disc (IVD) endplate in the different groups. Sox-9, Collagen type II, and Aggrecan genes in different groups of IVD endplate tissues were detected by qPCR. Vascular endothelial growth factor (VEGF), Wnt3α, β-catenin, glycogen synthase kinase-3β (GSK-3β), and vitamin D receptor (VDR) protein in IVD endplate tissues of different groups was detected by immunohistochemistry. IVD VDR protein and IDD-related protein levels were detected by Western blot. Linear trend tests and correlations were adopted to analyze the relationship between different degrees of IDD and VDR level. The results showed that the average particle size of the PVA-TGF-β1@PLGA NSs was approximately 415 nm, and Fourier infrared detection proved that PVA-TGF-β1@PLGA NSs were prepared. Sox-9, Collagen Type II, and Aggrecan gene levels in group M were notably inferior to those in group C (P &lt; 0.05). Sox-9, collagen type II, and aggrecan gene levels in group P were substantially increased versus group M (P &lt; 0.05). β-catenin level in groups P and M was greatly superior to that in group C (P &lt; 0.05). GSK-3β and VEGF levels in group P were increased relative to group M (P &lt; 0.05), while that of Wnt3α was decreased (P &lt; 0.05). Western blotting confirmed that there was a negative relationship between the expression of IDD-related proteins and VDR. The results showed that PLGA-coated ESNS could promote Collagen II expression in the matrix of the nucleus pulposus (NP), reduce proteoglycan loss, and promote cell proliferation and differentiation by regulating the Wnt/β-catenin signaling pathway (Wnt/β Sig). VDR level was negatively correlated with the development of IDD in rats.

The Analysis of Vitamin D Receptor Protein on Salmonella typhi infection in acute recurrent cases in endemic area in Eastern Indonesia

The host susceptibility mechanisms such as Vitamin D Receptor (VDR) is involved in the modulation of macrophage function and may possibly correlate with immunity disease including the severity of typhoid fever symptoms. The study aimed to assess the VDR Protein expression in the serums of recurrent acute typhoid fever (RATF) patients and compares it with typhoid fever (TF) patients, and healthy persons (HP). The study employed 30 RATF patients and 30 TF patients selected from primary health centres and hospitals in Eastern Indonesia as the endemic area. All the samples were obtained from several health centers in South Sulawesi, Southeast Sulawesi, Central Sulawesi, East Kalimantan and Papua and then collected in the sample bank Biology Laboratory of Molecular Immunology, Faculty of Medicine, Hasanuddin University. As a comparison, 30 samples of healthy persons were also selected from the Blood Transfusion Unit in Makassar, South Sulwesi Indonesia. The profile of VDR Protein was analyzed with Enzyme-linked Immunosorbent Assay (ELISA). VDR protein content data on RATF and TF were designed according to completely randomized design T test. Subsequently, it correlated to Pearson correlation to determine the interaction between Widal titre and VDR protein levels. A comparison between Widal titre and VDR Protein level was also made to identify the correlation. It was found that the mean of VDR protein expression of RATF was 13,44 ng/mL, the mean of VDR protein expression of TF was 24,88 ng/mL, and the mean of VDR protein expression of HP was 43,49 ng/mL. The correlation results between RATF-TF Widal titre and VDR protein level indicated a negative correlation with p-value of 0,004. There were significant differences in the VDR expression in the RATF, TF, and HP. RATF VDR expression lower than TF and HP and there was also a correlation between Widal titre with VDR Protein expression.

Placental mRNA and Protein Expression of VDR, CYP27B1 and CYP2R1 Genes Related to Vitamin D Metabolism in Preeclamptic Women

(1) Background: Considerable evidence indicates that the occurrence of preeclampsia (PE) is associated with a reduced vitamin D (VD) level. Several studies have found that VD deficiency is correlated with disturbed trophoblast invasion, reduced angiogenesis and increased vasoconstriction. Because the vitamin D receptor (VDR) and CYP27B1 and CYP2R1 hydrolases are strongly involved in VD metabolism, the goal of the present study was to evaluate their genes and proteins expression in the placentas from preeclamptic women. (2) Methods: Samples and clinical data were obtained from 100 Polish women (41 women with preeclampsia and 59 healthy pregnant controls). The whole PE group was divided into subgroups according to gestation week of pregnancy ending before and after 34 gestational weeks (early/late-onset preeclampsia (EOPE/LOPE)). However, finally, to reduce confounding by differences in gestational age, the EOPE group was excluded from the analysis of mRNA and protein placental expression, and we focus on the comparison between LOPE and control groups. The placental VDR, CYP27B1 and CYP2R1 mRNA expression was analyzed using RT-PCR, and placental protein levels were determined by ELISA assay. (3) Results. (3.1) Placental gene expression: Expression levels of both genes, CYP27B1 (1.17 vs. 1.05 in controls, p = 0.006) and CYP2R1 (2.01 vs. 1.89 in controls, p = 0.039), were significantly higher in preeclamptic placentas than in the control group. Interestingly, VDR expression was significantly lower in placentas from the PE group (1.15 vs. 1.20 in controls, p = 0.030). After dividing all preeclamptic women into subgroups only for the CYP27B1 gene, a significantly higher placental expression in the LOPE subgroup than the healthy controls was observed (padj = 0.038). (3.2) Placental protein expression: The results revealed that protein expression levels of CYP27B1 in the preeclamptic group were similar (5.32 vs. 5.23 in controls, p = 0.530). There was a significant difference in median VDR and CYP2R1 protein levels between studied groups (VDR: 2.56 vs. 3.32 in controls, p &lt; 0.001; CYP2R1: 1.32 vs. 1.43 in controls, p = 0.019). After stratification of preeclamptic women into subgroups, a significant difference was observed only in the VDR protein level. The medians in the LOPE subgroups were significantly lower compared to the healthy control group. In the whole study group, the placental VDR protein level was inversely correlated with systolic and diastolic blood pressure (all p &lt; 0.001), and positively correlated with gestational age (p &lt; 0.001) and infant birth weight (p = 0.014). (4) Conclusions: Lower mRNA and protein expression of VDR in preeclamptic placentas, and also VDR protein expression, could play a pivotal role in preeclampsia development. Additionally, the higher mRNA expression of both CYP27B1 and CYP2R1 hydrolase genes in placentas from preeclamptic women could indicate the compensatory role of these enzymes in preeclampsia etiology. Our results also indicate that placental VDR protein level could be one of the factors modulating blood pressure in pregnant women, as well as influencing gestational age and infant birth weight. Considering the importance of these findings, future studies are warranted.

Diagnostic Value of VDR in Bone Metastasis and Prognosis of Patients with Breast Cancer and Expression Correlation between Vitamin D Receptor and Hairless Protein

Background: Breast cancer is more likely to metastasize to the bone. Previous researches have revealed that the vitamin D receptor (VDR) contributes to breast cancer progression and bone metastasis in mouse and human breast cells, and hairless (Hr) protein interacts with VDR in the mammalian hair cycle. This study aimed to explore the expression of VDR/Hr in breast cancer, and the correlation between VDR/Hr and prognosis, bone metastasis, and metastasis-related prognosis. Methods: The expression of VDR and Hr was analyzed on 119 breast cancer tissues and corresponding normal breast tissue from each of the breast cancer samples by immunohistochemistry staining, and the databases were supplemented as well. Results: The expression of the VDR protein was significantly decreased in breast cancer patients (p < 0.05), inversely, the UALCAN (p = 0.000) and GEPIA (p > 0.05) databases showed that the VDR mRNA expression tended to be higher in tumor tissues. The Hr protein was expressed at a low level within breast cancer specimens (p < 0.05), which was in agreement with the level of Hr mRNA in UALCAN (p = 0.005) and GEPIA (p > 0.05). The protein levels of VDR and Hr were positively correlated (p > 0.05), while the mRNA levels suggested a close relationship with GEPIA (p < 0.05). Low expression of Hr protein displayed a tendency for longer overall survival (OS) and recurrence-free survival (RFS), and its mRNA data also revealed the same trend in the Kaplan-Meier dataset (both p > 0.05). However, VDR protein and mRNA with low expression had markedly shorter OS and RFS (both p < 0.05). The downregulation of VDR protein was significantly associated with an advanced stage (p < 0.05). Low VDR protein was an independent risk factor for poor prognosis (p < 0.05) and was negatively correlated with bone metastasis (p < 0.05). VDR protein and mRNA levels were both downregulated in breast cancer with bone metastasis (both p < 0.05). The area under ROC curve (AUC) for VDR protein expression to identify patients with bone metastasis was 0.661 (p < 0.05) and the AUC for VDR level to predict 1-year, 3-year, and 5-year OS was 0.621, 0.664, and 0.805 in patients with bone metastasis, respectively (p < 0.05). VDR with low expression accelerated bone metastasis and metastasis-related poor survival (both p < 0.05). Conclusion: VDR expression is a notable prognostic factor in primary breast cancer patients for predicting bone metastases and unfavorable clinical outcome.

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.

Association between vitamin D and ovarian cancer development in BRCA1 mutation carriers.

Objective: Women with inherited mutations in BRCA1 gene have a high (40–70%) genetic risk of developing ovarian cancer. Epidemiological studies suggest an inverse correlation between serum vitamin D (VD) levels and the risk of ovarian cancer, but there is a lack of data from BRCA1 mutation (BRCA1mut) carriers. Therefore, we investigated VD levels and actions in cancer free women with BRCA1 mutations.Materials and Methods: Blood, ovary and fallopian tube samples were collected from healthy pre-menopausal women with BRCA1mut and without BRCA1 mutations (BRCAwt). Serum calcifediol (major circulating form of VD) concentrations were measured by electrochemiluminescence immunoassay. Immunohistochemistry was performed on paraffin-embedded ovarian and fallopian tube sections to determine vitamin D receptor (VDR) expression. Ovarian surface epithelial cells (OSEs) from BRCA1mut carriers were cultured with or without calcitriol supplementation for 72 hrs. VDR protein levels, cell proliferation and cell viability were analyzed.Results: BRCA1mut women had lower serum calcifediol levels compared to BRCAwt women (p = 0.003). VDR protein expression was evident in ovarian and the fallopian tube epithelium of BRCAwt patients, but was reduced in BRCA1mut women. Calcitriol (biologically active VD) supplementation elevated VDR expression in cultured BRCA1mut OSEs (p = 0.005) and decreased cell proliferation rates in a dose-dependent manner without inducing apoptosis.Conclusions: VD biosynthesis and signaling via VDR in the ovarian and fallopian tube epithelium are impaired in BRCA1mut women. VD treatment may limit BRCA1mut epithelial cell proliferation without affecting cell viability, providing a rationale for exploring the potential for VD in ovarian cancer prevention in BRCA1mut carriers.

Vitamin D receptor is overexpressed in the duodenum of patients with irritable bowel syndrome.

Irritable bowel syndrome (IBS) is one of the most common functional gastrointestinal disorders, and bile acids are thought to be associated with the pathogenesis of IBS. Bile acid receptors are expressed on intestinal epithelial cells. However, no study has assessed bile acid receptor proteins in IBS. Therefore, we examined the intestinal mucosal expression of bile acid receptors in patients with IBS. Intestinal biopsies were performed in patients with IBS and controls. Mast cells, vitamin D receptor (VDR), and somatostatin were stained with specific antibodies. Levels of VDR, farnesoid X receptor (FXR), takeda-G-protein-receptor-5 (TGR5), claudins, and transient-receptor-potential-cation-channel-subfamily-V-member 6 (TRPV6) were assessed by western blotting. 3Mast cell counts in the second part of the duodenum were significantly higher in patients with IBS than in controls. VDR protein levels were significantly elevated in the duodenum and terminal ileum of patients with IBS compared with controls, although this difference was not seen in the cecum or rectum. FXR and TGR5 protein levels did not differ in any part of the intestine. VDR-positive cryptal epithelia in IBS were distributed not only at basal crypt but also along the upper part of the basal crypt epithelial cells. In contrast, the pattern of gut somatostatin-positive cells, claudins, and TRPV6 levels did not differ. The number of mast cells in the duodenum was significantly increased, and the protein expression levels of VDR, but not those of FXR or TGR5, were elevated in the duodenal epithelial crypt in patients with IBS.

The vitamin D receptor Taq I polymorphism is associated with reduced VDR and increased PDIA3 protein levels in human intestinal fibroblasts

The synonymous single nucleotide polymorphism (SNP) rs731236, located in the vitamin D receptor (VDR) gene (Taq I) has been associated with both decreased levels of the protein in peripheral blood mononuclear cells and a fibrosis-related complication in Crohn´s disease (CD). Interactions between VDR and a protein-disulfide isomerase-associated 3 (PDIA3) in the regulation of extracellular matrix have been reported and we aim to analyze the relevance of the VDR genotypes and the effects of Vitamin D (VD) in the expression of VDR, PDIA3 and proliferation of intestinal fibroblasts. Human intestinal fibroblasts were isolated from the non-affected surgical resections of colorectal patients and classified according to the VDR genotype. In some cases, cells were transfected with specific PDIA3 siRNA. Basal and VD-stimulated expression of VDR, PDIA3 and Collagen 1A1 (COL1A1) as well as fibroblast migration/proliferation were analyzed. Our data show that intestinal fibroblasts homozygous for the C allele in the VDR gene exhibited lower VDR protein levels and higher proliferation than cells homozygous for the T allele. VD increased VDR and attenuated the accelerated proliferation of CC fibroblasts. The diminished VDR level detected in CC cells was associated with increased levels of both PDIA3 and COL1A1 expression and the transient silencing of PDIA3 significantly reduced COL1A1 expression. We conclude that intestinal fibroblasts homozygous for the C allele in the VDR gene exhibited: reduced VDR protein levels, increased proliferation and increased PDIA3/COL1A1 expression. Treatment with VD increased VDR and attenuated proliferation of these cells.

Diminished Vitamin D Receptor Protein Levels in Crohn's Disease Fibroblasts: Effects of Vitamin D.

Vitamin D (VD) deficiency has been associated to Crohn’s disease (CD) pathogenesis, and the exogenous administration of VD improves the course of the disease, but the mechanistic basis of these observations remains unknown. Vitamin D receptor (VDR) mediates most of the biological functions of this hormone, and we aim to analyze here the expression of VDR in intestinal tissue, epithelial cells, and fibroblasts from CD patients. The effects of VD on a fibroblast wound healing assay and murine intestinal fibrosis are also analyzed. Our data show diminished VDR protein levels in surgical resections and epithelial cells from CD patients. In intestinal fibroblasts isolated from damaged tissue of CD patients, we detected enhanced migration and decreased VDR expression compared with both fibroblasts from non-damaged tissue of the same CD patient or control fibroblasts. Treatment with VD increased VDR protein levels, avoided the accelerated migration in CD fibroblasts, and prevented murine intestinal fibrosis induced by the heterotopic transplant model. In conclusion, our study demonstrates diminished VDR protein levels associated with enhanced migration in intestinal fibroblasts from damaged tissue of CD patients. In these cells, VD accumulates VDR and normalizes migration, which supports that CD patients would benefit from the VD anti-fibrotic therapeutic value that we demonstrate in a murine experimental model.

Characterization of VDR and CYP27B1 expression in the endometrium during the menstrual cycle before embryo transfer: implications for endometrial receptivity

BackgroundMolecular analyses of vitamin D in a typical cycling endometrium has received minimal research attention in the reproductive field. This study was designed to assess how expression of the endometrial vitamin D receptor (VDR) and CYP27B1, a vitamin D metabolizing enzyme, change during the menstrual cycle in women of reproductive age. In addition, this study explores the association between expression of vitamin D-VDR system and endometrial receptivity during the implantation window.MethodsSixteen patients underwent standardized in vitro fertilization (IVF) treatment and freeze-all techniques. Before embryo transfer, total serum 25(OH) D levels were determined through blood samples and VDR, CYP27B1, HOXA10, and CYP19 expression were determined through endometrial samples. Endometrial receptivity was also assessed using an electron microscope.ResultsWe found that VDR protein expression was significantly lower throughout the endometrial secretory phase compared to the proliferative phase, while CYP27B1 expression remained constant during the menstrual cycle. During the implantation window, ultrastructural evaluation showed that higher serum vitamin D levels were associated with more mature pinopodes; VDR and HOXA10 protein expression were substantially elevated in pregnant women compared to non-pregnant women; and VDR protein levels were positively correlated with HOXA10 levels. In addition, serum vitamin D levels were positively correlated with VDR and HOXA10 protein levels in the endometrium.ConclusionsWomen with increased VDR expression in the endometrium, especially during the implantation window of the menstrual cycle, were significantly more likely to be pregnant than women with decreased expression. Our results support the hypothesis that the Vitamin D-VDR system performs a role during the development of endometrial receptivity.

P046 Vitamin D decreases PDIA3 and prevents the enhanced migration of fibroblasts from stricturing Crohn’s disease

Abstract Background Fibrosis is a common complication in Crohn’s disease (CD) patients and fibroblasts play an important role in the fibrogenic process. Low vitamin D (VD) levels and a defective VD-signalling pathway have been reported in CD. VD signals through both vitamin D receptor (VDR) and protein disulfide-isomerase A3 (PDIA3) and we have previously demonstrated that VDR protein levels are reduced in fibroblasts isolated from CD patients and that VD increased VDR expression in these cells (A-2080; ECCO 2019). We aim to analyse here the effect of VD on both PDIA3 protein levels and migration in CD fibroblasts. Methods We used intestinal fibroblasts isolated from surgical resections of the damaged mucosa of CD patients with stricturing behaviour (B2). Control fibroblasts were obtained from the non-damaged intestine of patients with colorectal cancer. Fibroblasts were treated with VD (100 nM) or its vehicle for 24 h and PDIA3 protein levels were measured by Western Blot. In the wound healing analysis, a single scraping was done in the centre of the fibroblasts monolayer and FBS-free medium was added to the cells, which allows us to determine the ability of fibroblasts to migrate and close the wound. Photos were taken at 0, 24 and 48 h. Results of wound healing were expressed as the percentage of the wound at each time point for the maximal wounded area (time 0, 100%). Statistical significance was measured by ANOVA or t-test. Results No significant differences in PDIA3 protein levels were detected between control and CD fibroblasts but VD significantly decreased PDIA3 expression in CD fibroblasts (Figure 1A). In the wound healing assay, we detected that CD-B2 fibroblasts migrate faster than control cells, resulting in a reduced wounding area, 48 h later (Figure 1B). Treatment of these CD-B2 cells with VD decreased their migration rate, and 48 h later cells exhibited a higher and significant wounding area than vehicle-treated cells (Figure 1C). Conclusion Vitamin D decreased PDIA3 expression and prevented the accelerated migration detected in intestinal fibroblasts from B2-CD patients which suggest an anti-fibrotic effect of VD mediated by a direct effect of this hormone on intestinal fibroblasts.

Association of Fok1 VDR polymorphism with Vitamin D and its associated molecules in pulmonary tuberculosis patients and their household contacts

Status of Fok I VDR polymorphism along with vitamin D, Vitamin D receptor (VDR), and cathelicidin levels in Tuberculosis (TB) patients compared to household contacts and implication of these findings in susceptibility to TB is not known. 150 active TB patients, 150 household contacts and 150 healthy controls were recruited from North Indian population. Fok1 VDR polymorphism was studied by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP).VDR mRNA and protein levels were studied using quantitative real time PCR (q rt PCR) and enzyme linked immunosorbent assay (ELISA) respectively. Cathelicidin and Vitamin D levels were measured using ELISA and chemiluminescence immunoassay (CLIA) respectively. Significant association was found between Fok1 polymorphism and susceptibility to TB (P < 0.0005). VDR mRNA, VDR protein and vitamin D levels were significantly lower in active TB group when compared to household contacts and healthy controls (P < 0.0001, 0.0001 and 0.0005 respectively). Cathelicidin levels were higher in active TB patients compared to other groups (P < 0.0001). Expression of VDR and cathelicidin was significantly higher among ‘FF’ genotypes of VDR (more active form of VDR) compared to ‘ff’ genotype (less active form of VDR). ‘f’ allele was associated with increased susceptibility to TB. Higher frequency of ‘F’ allele, increased VDR expression along with increased vitamin D levels in household contacts compared to active TB group might be responsible for protection against active TB.

A negative feedback loop of H19/miR-675/VDR mediates therapeutic effect of cucurmin in the treatment of glioma.

Curcumin (CUR) shows a remarkable antitumor activity against a wide range of cancers such as glioma, but its underlying mechanism remains elusive. In this study, we aimed to explore the potential role of H19/miR-675/vitamin D receptor (VDR) in the effect of CUR against glioma. Real-time polymerase chain reaction and western-blot analysis were used to study the effect of CUR or 1,25-dihydroxyvitamin D (1,25(OH)2 D3 ) on the expression of H19, miR-675, and VDR. In addition, the effect of H19 on VDR expression was also studied. Furthermore, the expression of H19, miR-675, and VDR between CUR-loaded nanoparticles (NPs) and NP groups was compared, and the interaction among H19, miR-675, and VDR was analyzed by in-silicon and luciferase assays. In a dose-dependent manner, CUR and 1,25(OH)2 D3 both downregulated the expression of H19 and miR-675 but increased the expression of VDR. In addition, H19 evidently reduced the mRNA and protein levels of VDR. Furthermore, VDR was confirmed as a target gene of miR-675, which significantly reduced the expression of VDR. Finally, the administration of CUR evidently decreased tumor volume. CUR-loaded NP group exhibited lower levels of H19 and miR-675, while the NP group showed higher levels of VDR mRNA and protein. In summary, it is the first time that the involvement of a negative feedback loop of H19/miR-675/VDR has been demonstrated in the development of glioma. Therefore, H19 might serve as a new biomarker for the diagnosis and treatment of glioma.