Vitamin D Receptor Protein Expression Research Articles

Association between vitamin D receptor polymorphisms, tight junction proteins and clinical features of adult patients with atopic dermatitis

Background: Few studies have explored the intricate connections between vitamin D receptor (VDR) gene polymorphisms, VDR, tight junction (TJ) protein expression and clinical features of atopic dermatitis (AD). Methods: From 43 adult AD patients, VDR polymorphisms were genotyped from peripheral blood samples using polymerase chain reaction-restriction fragment length polymorphism. VDR, occludin, claudin-1 and ZO-1 protein expression from skin lesion biopsies were assessed by immunohistochemistry. Results: The A1012G heterozygous VDR polymorphism exhibited a lower odds ratio (OR) for juvenile AD onset (OR: 0.046, 95% CI 0.004-0.51, p=0.012). In contrast, the presence of ≥2 homozygous VDR polymorphisms were significantly associated with positive skin prick test (SPT) (10/20, 50%) vs. negative SPT (1/23, 4.3%; p=0.0003). The most highly expressed TJ proteins in lesions of AD patients were claudin-1 and ZO-1, while VDR and occludin were less prevalent. A significant correlation was observed between ZO-1 expression and a body mass index ≥30 kg/m2 (OR: 12.1, 95% CI 1.06-137.9, p=0.045). Claudin-1 expression was associated with a positive SPT (OR: 8.23, 95% CI 1.04-65.5, p=0.046) and serum 25(OH)D levels were negatively correlated with ZO-1 expression (rho= -0.43, p=0.0058). Conclusion: This study provides novel insights into the relationship between VDR gene polymorphisms, VDR, TJ protein expression, and clinical features in adult AD patients, highlighting a significant role of vitamin D in the pathophysiology of this disease.

Effect of exogenous calcitriol on myopia development and axial length in guinea pigs with form deprivation myopia

The annual increase in myopia prevalence poses a significant economic and health challenge. Our study investigated the effect of calcitriol role in myopia by inducing the condition in guinea pigs through form deprivation for four weeks. Untargeted metabolomics methods were used to analyze the differences in metabolites in the vitreous body, and the expression of vitamin D receptor (VDR) in the retina was detected. Following form deprivation, the guinea pigs received intraperitoneal injections of calcitriol at different concentrations. We assessed myopia progression using diopter measurements and biometric analysis after four weeks. Results indicated that form deprivation led to a pronounced shift towards myopia, characterized by reduced choroidal and scleral thickness, disorganized collagen fibers, and decreased scleral collagen fiber diameter. Notably, a reduction in calcitriol expression in vitreous body, diminished vitamin D and calcitriol levels in the blood, and decreased VDR protein expression in retinal tissues were observed in myopic guinea pigs. Calcitriol administration effectively slowed myopia progression, preserved choroidal and scleral thickness, and prevented the reduction of scleral collagen fiber diameter. Our findings highlight a significant decrease in calcitriol and VDR expressions in myopic guinea pigs and demonstrate that exogenous calcitriol supplementation can halt myopia development, enhancing choroidal and scleral thickness and scleral collagen fiber diameter.

Roles of Glutathione and AP-1 in the Enhancement of Vitamin D-Induced Differentiation by Activators of the Nrf2 Signaling Pathway in Acute Myeloid Leukemia Cells.

Active vitamin D derivatives (VDDs)-1α,25-dihydroxyvitamin D3/D2 and their synthetic analogs-are well-known inducers of cell maturation with the potential for differentiation therapy of acute myeloid leukemia (AML). However, their dose-limiting calcemic activity is a significant obstacle to using VDDs as an anticancer treatment. We have shown that different activators of the NF-E2-related factor-2/Antioxidant Response Element (Nrf2/ARE) signaling pathway, such as the phenolic antioxidant carnosic acid (CA) or the multiple sclerosis drug monomethyl fumarate (MMF), synergistically enhance the antileukemic effects of various VDDs applied at low concentrations in vitro and in vivo. This study aimed to investigate whether glutathione, the major cellular antioxidant and the product of the Nrf2/ARE pathway, can mediate the Nrf2-dependent differentiation-enhancing activity of CA and MMF in HL60 human AML cells. We report that glutathione depletion using L-buthionine sulfoximine attenuated the enhancing effects of both Nrf2 activators concomitant with downregulating vitamin D receptor (VDR) target genes and the activator protein-1 (AP-1) family protein c-Jun levels and phosphorylation. On the other hand, adding reduced glutathione ethyl ester to dominant negative Nrf2-expressing cells restored both the suppressed differentiation responses and the downregulated expression of VDR protein, VDR target genes, as well as c-Jun and P-c-Jun levels. Finally, using the transcription factor decoy strategy, we demonstrated that AP-1 is necessary for the enhancement by CA and MMF of 1α,25-dihydroxyvitamin D3-induced VDR and RXRα protein expression, transactivation of the vitamin D response element, and cell differentiation. Collectively, our findings suggest that glutathione mediates, at least in part, the potentiating effect of Nrf2 activators on VDDs-induced differentiation of AML cells, likely through the positive regulation of AP-1.

Association Between Expression of Vitamin D Receptor and Insulin-Like Growth Factor 1 Receptor Among Breast Cancer Patients.

Vitamin D receptor (VDR) and insulin-like growth factor 1 receptor (IGF1R) are known to be involved in breast cancer (BC) progression. Our previous work reported a correlation of differential localization of IGF1R with hormone receptor status in BC. A recent report described VDR and IGF1R as potential indicators of BC prognosis, but their interplay was not discussed. The present study focused on understanding the association of VDR expression with IGF1R activation, different molecular markers, and subtypes of BC. A retrospective study was designed to evaluate the VDR expression among 48 BC patients pathologically diagnosed as invasive BC and were surgically treated at Sharjah Breast Care Center, University Hospital Sharjah (UHS), United Arab Emirates (UAE). Formalin-fixed paraffin-embedded (FFPE) tumor blocks with appropriate clinicopathological data were subjected to immunohistochemistry (IHC), and VDR protein expression was interpreted based on the staining intensity (SI) and the percentage of the positively stained cells (PP). Nearly 44% of cases in the study were vitamin D deficient. A positive VDR expression with strong intensity (score > 4) was seen in 27 cases (56.3%). The expression pattern for VDR was equally distributed in cytoplasm and nucleus. For the IGF1R intensity, 24 cases (50%) of total cohort showed strong expression. A significant association was detected between IGF1R and VDR expression (P = 0.031). The present study identified positive association between IGF1R and VDR expression where most of the cases with strong VDR expression displayed strong IGF1R expression. These findings may contribute to current understanding on the role of VDR in BC and its interaction with IGF1R.

Aerobic Exercise Ameliorates Myocardial Fibrosis via Affecting Vitamin D Receptor and Transforming Growth Factor-β1 Signaling in Vitamin D-Deficient Mice.

Myocardial fibrosis is a pathological phenomenon associated with cardiovascular disease (CVD) that plays a crucial role in the development of heart diseases. Vitamin D deficiency can promote the development of CVD and exercise plays a role in the treatment of CVD. This study aimed to explore the effects of 12-week aerobic exercise training on myocardial fibrosis in vitamin D-deficient mice. A vitamin D-deficient mouse model was induced by a vitamin D-deficient (0 IU Vitamin D3/kg) diet. Twenty-four C57BL/6J male mice were randomly divided into three groups: a control sedentary group (CONS, n = 8), a vitamin D-deficient sedentary group (VDDS, n = 8), and a vitamin D-deficient exercise group (VDDE, n = 8) which was aerobically trained for 12 weeks. The results showed that the serum 25-hydroxyvitamin D [25(OH)D] levels of the VDDS group were <50 nmol/L, which was significantly lower than that of the CONS group. Compared with the CONS group, the VDDS group showed cardiac dysfunction and significant fibrosis, together with lower vitamin D receptor (VDR) mRNA and protein expression levels, higher mRNA expression levels of profibrotic and inflammatory factors, and higher transforming growth factor-β1 (TGF-β1) and phospho-Smad2/3 (P-Smad2/3) protein expression levels. Serum 25(OH)D levels in the VDDE group were significantly higher than those in the VDDS group. Compared with the VDDS group, the VDDE group showed improved cardiac function and alleviated myocardial fibrosis. Meanwhile, the VDDE group had significantly higher VDR mRNA and protein expression levels; lower mRNA expression levels of profibrotic and inflammatory factors; and lower TGF-β1 and P-Smad2/3 protein expression levels. In conclusion, aerobic exercise training remains a promising intervention for treating myocardial fibrosis in vitamin D deficiency.

Bone Metastases of Diverse Primary Origin Frequently Express the VDR (Vitamin D Receptor) and CYP24A1.

Active vitamin D (1,25(OH)2D3) is known to exert direct anti-cancer actions on various malignant tissues through binding to the vitamin D receptor (VDR). These effects have been demonstrated in breast, prostate, renal and thyroid cancers, which all have a high propensity to metastasise to bone. In addition, there is evidence that vitamin D catabolism via 24-hydroxylase (CYP24A1) is altered in tumour cells, thus, reducing local active vitamin D levels in cancer cells. The aim of this study was to assess VDR and CYP24A1 expression in various types of bone metastases by using immunohistochemistry. Overall, a high total VDR protein expression was detected in 59% of cases (39/66). There was a non-significant trend of high-grade tumours towards the low nuclear VDR expression (p = 0.07). Notably, patients with further distant metastases had a reduced nuclear VDR expression (p = 0.03). Furthermore, a high CYP24A1 expression was detected in 59% (39/66) of bone metastases. There was a significant positive correlation between nuclear VDR and CYP24A1 expression (p = 0.001). Collectively, the VDR and CYP24A1 were widely expressed in a multitude of bone metastases, pointing to a potential role of vitamin D signalling in cancer progression. This is of high clinical relevance, as vitamin D deficiency is frequent in patients with bone metastases.

Expression of Vitamin D Receptor (VDR) in Urinary Bladder Carcinoma: Immunohistochemical and Histopathological Study

Background: Bladder cancer is the most common malignancy of the urinary tract. Calcitriol [1,25 (OH)2vitamin D3] has anticancer effects mediated through binding to vitamin D receptor (VDR). The expression of VDR is present in many normal and cancer tissues. But, there is little information about its expression in urinary bladder carcinoma. This study aimed to analyze VDR immunohistochemical expression in 74 Egyptian patients with urinary bladder carcinoma and to evaluate its association with different clinicopathological parameters. Methods: Sections from formalin-fixed, paraffin-embedded tumor blocks were stained immunohistochemically using monoclonal anti-VDR antibody. VDR protein expression as well as its immunostaining patterns were recorded and scored separately in each case using semi-quantitative immunoreactive score. Results: VDR was consistently expressed in the included histologically normal urothelium while tumor cells showed variable degrees of expression. Cytoplasmic/membranous VDR expression was common among the studied cases especially those with urothelial morphology (p = 0.076). While, the mean nuclear VDR was significantly (p = 0.007) higher in non-urothelial tumors. Nuclear VDR was significantly associated with muscle invasion (p = 0.000) and tumor stage (p = 0.001) in urothelial carcinoma. It was also statistically related to tumor grade, stage and muscle invasion in non-urothelial tumors (p = 0.002, 0.003 and 0.012, respectively). Conclusion: there was a significant relation between nuclear VDR expression and prognostic markers suggesting its decrease as an indicator of a poorer prognosis. Vitamin D supplementation may represent a new treatment option for patients with bladder cancer.

Upregulation Of Vitamin D Pathways Is Inversely Associated With Quadriceps Fiber Atrophy Following ACL Tear.

Protracted quadriceps atrophy and weakness after Anterior Cruciate Ligament (ACL) injury results in poor knee mechanics and contributes significantly to the development of posttraumatic osteoarthritis. The cellular and molecular underpinnings of poor functional recovery are unknown, limiting the development of evidence-based therapies. Vitamin D is a fat-soluble vitamin with critical roles for bone and skeletal muscle health. PURPOSE: Our goal is to determine how participants’ skeletal muscle expression of vitamin D receptor (VDR), CYP24A1, and CYP27B1 is affected by anterior cruciate ligament (ACL) tear. METHODS: Fifteen participants (mean ± SD; age, 22.3 ± 5.4; BMI, 27 ± 5; 47% female) who sustained an ACL tear received muscle biopsies from the injured and noninjured vastus lateralis. Muscle biopsies were processed for western blotting and immunohistochemistry. The expression of VDR, CYP24A1, and CYP27B1 were assayed via western blot. Seven μm sections were cut on a cryostat and fiber cross-sectional area (CSA) was assessed with automated methods. Arbitrary densitometry units (AU) from western blotting and mean fiber CSA results were compared between limbs using paired t-tests. Pearson correlations were used to identify associations between VDR protein expression in the injured limb and pooled muscle fiber CSA. RESULTS: VDR expression was 57% higher in the injured limb when compared to the noninjured limb (0.11 ± 0.05 vs 0.07 ± 0.037 AU, p = 0.024). The expression of CYP24A1 were CYP27B1 were higher in the injured limb, but the differences did not reach statistical significance. Additionally, mean fiber CSA was lower in the injured limb (4259 ± 990 vs. 4976 ± 1115 μm2, p = 0.005). VDR expression in the injured limb and VDR percent difference between limbs was inversely associated with fiber atrophy (as assessed as CSA percent difference) (R = -0.575, p = 0.025; R = -0.545, p = 0.036). CONCLUSIONS: ACL injury increases VDR expression in quadriceps, and preliminary results suggest alterations in vitamin D activity and turnover. Our results support a role for vitamin D receptor expression to maintain quadriceps fiber size and highlight the need for further research into micronutrient metabolism to aid in the recovery from injury.

Vitamin D Receptor and Its Influence on Multiple Sclerosis Risk and Severity: From Gene Polymorphisms to Protein Expression

Multiple sclerosis (MS) is a multifactorial neurodegenerative disease. Low levels of vitamin D are a risk factor for MS and alterations in the vitamin D receptor (VDR) might be a risk factor as well. This study aimed to evaluate whether the VDR rs731236 (Taq-I) and rs4334089 (HpyCH4V) gene polymorphisms and VDR protein expression are associated with MS risk and severity. Vitamin D plasma levels were analyzed in a group of patients. Additional analyses of VDR protein expression and vitamin D levels of patients with different forms of MS (MSSS &lt; 3 and MSSS ≥ 3) were performed. The analysis of the genotypic and allelic frequencies revealed that the rs731236 (Taq-I) gene polymorphism is significantly associated with MS presence. Although the total, cytosolic and nuclear VDR protein contents do not change between MS patients and healthy controls and between patients with different MS severity, vitamin D levels decrease in parallel with an increase in MSSS.

Vitamin D Receptor/Vitamin D Response Element Directly Modulate Nestin Transcription to Ameliorate PAN-Induced Podocyte Morphological Changes

Background: The change of podocyte morphology is a pathologic feature of chronic kidney disease. Several studies have suggested that vitamin D plays a role in the protection of podocytes, but the underlying mechanism remains unclear. Methods: The effects of paricalcitol on podocyte injury were tested in a puromycin aminonucleoside (PAN)-induced rat model and cultured mouse podocytes. Proteinuria, podocyte foot process (FP) effacement, and the expression of nestin and vitamin D receptor (VDR) were evaluated. VDR-siRNA or plasmids containing VDR-shRNA were transfected into podocytes to silence VDR expression. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were performed to verify the connection between VDR and nestin gene expression. Results: Paricalcitol significantly alleviated proteinuria and podocyte FP effacement in PAN-induced nephrosis, which was accompanied by increased VDR expression in the glomeruli. Paricalcitol also inhibited PAN-induced nestin overexpression in the glomeruli. In an in vivo study, PAN significantly inhibited VDR protein expression, stimulated nestin protein expression, and resulted in nestin filament derangement in mouse podocytes, while paricalcitol treatment abolished these effects. In contrast, downregulation of VDR resulted in derangement and overexpression of nestin. ChIP assays demonstrated the presence of a vitamin D response element (VDRE) in the nestin promoter, and paricalcitol enhanced the binding of VDR to VDRE. Furthermore, luciferase reporter assays of the nestin promoter fragment showed that paricalcitol effectively repressed nestin reporter gene expression after PAN treatment, and mutation of VDRE abolished this effect. Conclusions: Paricalcitol directly regulates nestin transcription through the interaction of VDR/VDRE, thereby preventing morphological changes of podocytes in PAN nephropathy.

Vitamin D Receptor Activation Exerts Anti‐Secretory Actions In Colonic Epithelial Cells

BackgroundThe vitamin D receptor (VDR) is a nuclear receptor that is expressed in many tissues throughout the body, but is particularly abundant within the colon. VDR is classically known to be activated by the active form of vitamin D, calcitriol, but is appreciated to also serve as a receptor for luminal bile acids. VDR has previously been shown to have anti‐inflammatory and barrier promoting actions in the large intestine. However, there is little known of the role VDR plays in regulating colonic epithelial transport function. Interestingly, previous studies from our laboratory have shown the nuclear bile acid receptor, farnesoid X receptor (FXR), to exert anti‐secretory actions in colonic epithelial cells. The current study aims to investigate a potential role for VDR in regulating colonic Cl‐ secretion and whether it may be a target for the treatment of diarrhoeal diseases.AimTo investigate the effects of VDR activation by calcitriol on colonic epithelial Cl‐ secretion.MethodsPolarised monolayers of T84 cells, grown on permeable supports, were used as a model of the colonic epithelium. Protein expression of VDR and CFTR was measured by western blotting. Calcitriol (1 ‐ 20 nM) was used to activate VDR and the VDR target protein, CYP24A1, was used to verify activation of the receptor by qRT‐PCR. The specificity of calcitriol as a VDR agonist was determined by investigating FXR activation using the FXR target protein, FGF19, and a FXR‐luciferase reporter cell line. The effects of VDR activation by calcitriol (10 nM) on chloride secretion over a timecourse (6 – 48 hr) was determined using the Ussing chamber technique. Changes in short‐circuit current (Isc) induced by the Ca2+‐dependent agonist, carbachol (CCh; 100 µM) were assessed.ResultsVDR was found to be expressed in T84 cell monolayers as determined by western blotting. Treatment of T84 cells with calcitriol (5 ‐ 20 nM) significantly (n = 3, *p ≤ 0.05) increased expression of CYP24A1, indicating activation of VDR by the agonist. Calcitriol did not alter the activity of FXR, either in an FXR‐luciferase reporter cell line or in T84 cells, indicating specificity of the agonist for VDR. In Ussing chambers, the FXR agonist, GW4064 (5 µM; 24 hrs), inhibited Cl‐ secretory responses to CCh, as previously reported (n = 5, **p ≤ 0.01). Calcitriol (10 nM) was also found to significantly inhibit CCh‐stimulated Cl‐ secretory responses by 39.9 ± 5.1% (6 hr), 40.4 ± 4.3% (18 hr) and 62.0 ± 6.1% (24 hr) (n = 5, **p ≤ 0.01). In addition to their effects on CCh‐induced chloride secretion, GW4064 (5 µM; 24 hrs) downregulated the protein expression of CFTR by 0.5 ± 0.1 (n = 8, ***p ≤ 0.001) (as previously reported) and calcitriol (10 nM) downregulated CFTR by 0.35 ± 0.1 (n=8, *p≤ 0.05).ConclusionSimilar to the bile acid receptor, FXR, VDR is expressed in colonic epithelial cells where activation by its natural ligand, calcitriol, downregulates CFTR expression and inhibits agonist‐induced Cl‐ secretory responses. Since Cl‐ secretion is the primary driving force for intestinal fluid secretion, our data suggest that VDR may serve as a target for the development of new anti‐diarrhoeal agents. Future studies will aim to determine the molecular mechanisms involved and investigate a possible role for VDR in mediating anti‐secretory actions of luminal bile acids.

Identification of microRNAs targeting vitamin D receptor and their effect on parathyroid hormone secretion in secondary hyperparathyroidism

To identify the miRNAs targeting vitamin D receptor (VDR) gene and their effect on parathyroid hormone (PTH) secretion in secondary hyperparathyroidism. Primary parathyroid cells with secondary hyperparathyroidism were isolated by collagenase digestion and cultured. The miRNAs targeting VDR were screened by bioinformatics methods and full transcriptome sequencing, and dual-luciferase reporter assay was used to verify the targeting relationship between VDR and the screened miRNA. The effects of overexpression or inhibition of the candidate miRNA on VDR mRNA and protein expressions and PTH secretion were evaluated using qRT-PCR and Western blotting. The expression levels of the candidate miRNAs and VDR mRNA in clinical specimens of parathyroid tissues were verified by qRT-PCR, and the expression of VDR protein was detected by immunohistochemistry. We successfully isolated primary parathyroid cells. Dual-luciferase reporter assay verified the targeting relationship of hsa-miR-149-5p, hsa-miR-221-5p, hsa-miR-222-3p, hsa-miR-29a-5p, hsa-miR-301a-5p, hsa-miR-873-5p, hsa-miR-93-3p with VDR, and among them, the overexpression of hsa-miR-149-5p and hsa-miR-301a-5p significantly increased PTH secretion in the parathyroid cells. In patients with secondary hyperparathyroidism, hsa-miR-149-5p was highly expressed in the parathyroid tissues (P=0.046), where the expressions of VDR mRNA (P=0.0267) and protein were both decreased. The two miRNAs, hsa-miR-149-5p and hsa-miR-301a-5p, may promote the secretion of PTH in patients with secondary hyperparathyroidism by down-regulating the expression of VDR gene.

EB1089 promotes the expression of vitamin D receptor in the intestinal epithelial cell line HT-29 and reduces lipopolysaccharide-induced inflammatory response.

BackgroundEB1089 is a vitamin D receptor (VDR) agonist that reduces the inflammatory response. The specific pathway through which the inflammatory response is reduced is unclear, and this study is intended to investigate its effect on the inflammatory response through an LPS (lipopolysaccharide)-induced inflammation model of the intestinal epithelial cell line HT-29.MethodsWe divided HT-29 cells into a control group, LPS group, and LPS + EB1089 group. Cell proliferation was detected with a Cell Counting Kit-8 (CCK-8) kit, and the apoptosis rate was determined through flow cytometry, the lactate dehydrogenase (LDH), interleukin-18 (IL-18), and interleukin-1β (IL-1β) contents were measured with enzyme-linked immunosorbent assay (ELISA), and the expression levels of ASC, Caspase-1, NLRP3, VDR, TLR4, MYD88, NF-κB and other proteins in the cells were detected by western blot.ResultsPretreatment with EB1089 pretreatment reduced the LPS-induced apoptosis of HT-29 cells. LDH content was evidently lower in the LPS + EB1089 group than in the LPS group. The LDH content in the LPS + EB1089 group was evidently lower than that in the LPS group. The protein expression levels of ASC, Caspase-1, NLRP3, TLR4, MyD88, and NF-κB in the LPS group were evidently increased compared with those in the control group; however, the protein expression of VDR evidently decreased. The protein expression levels of Caspase-1, NLRP3, TLR4, MyD88, and NF-κB in the LPS + EB1089 group were evidently lower than those in the LPS group; however, the VDR protein expression evidently increased in the LPS + EB1089 group.ConclusionsEB1089 promoted the VDR expression and reduced the LPS induced inflammation in HT-29 cells.

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.

Tanshinone IIA attenuates high glucose-induced epithelial-to-mesenchymal transition in HK-2 cells through VDR/Wnt/β-catenin signaling pathway.

The progression of diabetic kidney disease (DKD) is closely related to renal tubular epithelial- to-mesenchymal transition (EMT) and tubulointerstitial fibrosis. Tanshinone IIA (TSIIA), extracted from a traditional Chinese medicine named Salvia miltiorrhiza, has been proved to have anti-fibrosis effects. The aim of this study was to investigate the effect of TSIIA on high glucose-induced EMT in human proximal tubular cells (HK-2 cells) and its possible mechanism. The proliferation of cells exposed to different concentrations of glucose was measured by light microscopy and CCK-8 test. The cells were stimulated with 30 mM glucose and different concentrations of TSIIA (5 μM or 10 μM) for 48 h. Vitamin D receptor (VDR)-siRNA was used to transfect cells, and high glucose and TSIIA treatment were further used to treat cells. The expression of alpha smooth muscle actin (a-SMA) mRNA was detected by qPCR to ensure successful induction of EMT, and the expression of VDR mRNA was detected by qPCR to ensure successful transfection of VDR-siRNA. Protein expression of a-SMA, E-cadherin, VDR, b-catenin and glycogen synthase kinase 3b (GSK-3b) was detected by Western blot analysis. The results showed that high glucose concentration inhibited cell proliferation and promoted EMT in HK-2 cells. TSIIA could reverse high glucose-induced EMT by increasing the level of VDR protein and inhibiting the levels of b-catenin and GSK-3b proteins suggestive of a negative correlation between VDR and the Wnt/b-catenin pathway. After VDR-siRNA transfection and incubation of cells at high glucose concentration, the inhibitory effect of VDR on the expression of b-catenin and GSK-3b of Wnt pathway was suppressed and the b-catenin pathway was activated. When VDR level was restored by TSIIA, the inhibitory effect of VDR on the pathway was also restored and the activation of the pathway was suppressed. TSIIA was able to attenuate high glucose-induced EMT in HK-2 cells by up-regulating VDR levels, which might be related to the inhibitory effect of VDR on the Wnt pathway.

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.

Cholecalciferol Mediates Apoptosis in Siha Cervical Cancer Line via Autocrine Mechanisms

Cervical cancer disproportionately affects low-resource countries and is a significant health burden in South Africa. Pre-clinical studies have demonstrated numerous anti-cancer actions of vitamin D metabolites. Here, the anti-cancer action of the vitamin D precursor, cholecalciferol, was investigated in a high-grade cervical cancer cell line, SiHa. SiHa cell cultures were treated with a range of cholecalciferol doses (26 nM, 104 nM, 260 nM and 2600 nM) for 72 hours. Cell count and viability were assessed by crystal violet and trypan blue assays, respectively. Apoptotic cell death was investigated by flow cytometry, which measured mitochondrial membrane potential (∆Ψm), phosphatidylserine (PS) externalisation, effector caspase activation and the expression of DNA damage markers. Additionally, brightfield microscopy and transmission electron microscopy (TEM) were respectively used to characterise morphological and ultrastructural features of apoptosis. Expression of the vitamin D metabolising system (VDMS) – consisting of cholecalciferol activating (CYP2R1 and CYP27A1), calcidiol activating (CYP27B1) and calcidiol inactivating (CYP24A1) enzymes, and the vitamin D receptor (VDR) – was assessed by qPCR and Western blots. Data were analysed using a one-way ANOVA and Bonferroni post-hoc tests and p < 0.05 was considered statistically significant. Significant decreases in cell count (p = 0.011) and cell viability (p < 0.0001) were identified in SiHa cells treated with 2600 nM cholecalciferol. Furthermore, biochemical markers at 2600 nM treatment were significant for apoptosis, and included decreased ∆Ψm (p = 0.0145); increased PS externalisation (p = 0.0439); terminal caspase activation (p = 0.0025); and nuclear damage (p = 0.004). Moreover, biochemical apoptosis was corroborated by classical apoptotic features observed by brightfield microscopy and TEM. Additionally, a significant increase in CYP2R1 gene (p < 0.0001) and protein (p = 0.021) expression, and a converse significant decrease in CYP27B1 gene (p = 0.003) and protein expression (p = 0.031) were observed at 2600 nM cholecalciferol treatment. Furthermore, significant increases in VDR gene (p = 0.033) and protein (p = 0.04) expression, and CYP24A1 gene (p < 0.0001) and protein (p = 0.0274) expression were observed at 2600 nM cholecalciferol. In summary, high-dose cholecalciferol treatment of SiHa cervical cancer cells inhibits cell growth, induces apoptosis, and furthermore, upregulates CYP2R1 and VDR expression. Taken together, these findings suggest that autocrine activation of cholecalciferol to calcidiol may mediate VDR signalling of cell growth inhibition, and apoptosis in SiHa experimental cultures.

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.

A single bout of high-intensity exercise modulates the expression of vitamin D receptor and vitamin D-metabolising enzymes in horse skeletal muscle.

The expressions of vitamin D receptor (VDR) and vitamin D-metabolising enzymes (CYP27B1 and CYP24A1) in skeletal muscle have been reported. However, the regulation of this vitamin D system in horse skeletal muscle after high-intensity exercise has not yet been elucidated. To investigate the effect of high-intensity exercise on the expression of vitamin D system-related proteins in horse skeletal muscle and its associations with skeletal muscle stem cell (SMSC) activity and serum 25(OH)D level. Longitudinal study. Six healthy ponies (5 geldings, 1 mare; age 6.3±2.2years) were studied. Serum and muscle samples were taken from the jugular vein and gluteus medius respectively. Samples were collected at pre-exercise, post-exercise, 1 and 3weeks after a single bout of high-intensity exercise. Protein expression levels of VDR, CYP27B1, CYP24A1, OxPhos and Pax7 (SMSC marker) were determined using immunohistochemical analysis. Oxidative capacity and intramuscular glycogen content were evaluated using histochemical analysis. Blood biochemistry was analysed for lactate concentration and creatine kinase (CK), and 25(OH)D activity. High-intensity exercise significantly upregulated Pax7 and VDR protein expression, which correlated with significantly increased blood lactate and serum CK levels immediately post-exercise. Serum 25(OH)D2 level correlated with CYP27B1 protein expression in skeletal muscle, and it reduced significantly immediately post-exercise and at 1 and 3weeks post-exercise. However, CYP24A1 protein expression was unchanged throughout study periods. The healthy ponies could not represent a fit population of racehorses and eventers. The rapid increase in Pax7 and VDR protein expression along with serum CK level after high-intensity exercise demonstrated an association between SMSC activity and activation of the vitamin D system in response to muscle injury in horses. Moreover, a decrease in CYP27B1 protein expression, correlated with a reduction in serum 25(OH)D2 , may indicate a compromised vitamin D metabolism after high-intensity exercise.