The Distinct Regulation of the Vitamin D and Aryl Hydrocarbon Receptors in COVID-19.

(1) Background: SARS-CoV-2 affects several immune pathways, including the vitamin D (VDR) and the aryl hydrocarbon receptor pathways (AhR). The aim of the study was the evaluation of the VDR and AhR pathways in the blood of COVID-19 patients with regard to the severity of disease. (2) Methods: Observational, single-center, case-control design. A total of 240 samples were selected for exploration. Patients who tested negative for SARS-CoV-2 but suffered from other respiratory infections (ORIs) served as a control group. (3) Results: VDR-specific mRNA in the blood of patients with mild symptoms (131.2 ± 198.6) was significantly upregulated relative to the VDR expression of the ORI group (23.24 ± 42.60; p < 0.0001); however, VDR expression of critically ill patients showed an impaired upregulation (54.73 ± 68.34; p < 0.001). CYP27B1 expression was not significantly regulated during SARS-CoV-2 infection. There was a downregulation of VDR and CYP27B1 compared to survivors. There was no significant difference in 25(OH)-vitamin D3 levels between critically ill patients with regard to survival (24.3 ± 9.4 vs. 27.1 ± 11.3; p = 0.433). (4) Conclusion: The VDR and AhR pathways are distinctively regulated in patients suffering from COVID-19 depending on the severity of disease. A combination treatment of antiviral drugs and vitamin D substitution should be evaluated for potentially improved prognosis in COVID-19.

Oleanolic acid exerts bone anabolic effects via activation of osteoblastic 25-hydroxyvitamin D 1-alpha hydroxylase.

Oleanolic acid (OA) is previously shown to exert bone protective effects in aged animals. However, its role in regulating osteoblastic vitamin D bioactivation, which is one of major causes of age-related bone loss, remains unclear. Our results revealed that treatment of OA significantly increased skeletal CYP27B1 expression and circulating 1,25(OH)2D3 in ovariectomized mice (p <0.01). Moreover, OA upregulated CYP27B1 protein expression and activity, as well as the vitamin D-responsive bone markers alkaline phosphatase (ALP) activity and osteopontin (OPN) protein expression, in human osteoblast-like MG-63 cells (p<0.05). CYP27B1 expression increased along with the osteoblastic differentiation of human bone marrow derived mesenchymal stem cells (hMSCs). CYP27B1 expression and cellular 1,25(OH)2D3 production were further potentiated by OA in cells at mature osteogenic stages. Notably, our study suggested that the osteogenic actions of OA were CYP27B1 dependent. In summary, the bone protective effects of OA were associated with the induction of CYP27B1 activity and expression in bone tissues and osteoblastic lineages. Hence, OA might be a potential approach for management of age-related bone loss.

Novel Vitamin D3 Hydroxymetabolites Require Involvement of the Vitamin D Receptor or Retinoic Acid-Related Orphan Receptors for Their Antifibrogenic Activities in Human Fibroblasts.

We investigated multiple signaling pathways activated by CYP11A1-derived vitamin D3 hydroxymetabolites in human skin fibroblasts by assessing the actions of these molecules on their cognate receptors and by investigating the role of CYP27B1 in their biological activities. The actions of 20(OH)D3, 20,23(OH)2D3, 1,20(OH)2D3 and 1,20,23(OH)3D3 were compared to those of classical 1,25(OH)2D3. This was undertaken using wild type (WT) fibroblasts, as well as cells with VDR, RORs, or CYP27B1 genes knocked down with siRNA. Vitamin D3 hydroxymetabolites had an inhibitory effect on the proliferation of WT cells, but this effect was abrogated in cells with silenced VDR or RORs. The collagen expression by WT cells was reduced upon secosteroid treatment. This effect was reversed in cells where VDR or RORs were knocked down where the inhibition of collagen production and the expression of anti-fibrotic genes in response to the hydroxymetabolites was abrogated, along with ablation of their anti-inflammatory action. The knockdown of CYP27B1 did not change the effect of either 20(OH)D3 or 20,23(OH)2D3, indicating that their actions are independent of 1α-hydroxylation. In conclusion, the expression of the VDR and/or RORα/γ receptors in fibroblasts is necessary for the inhibition of both the proliferation and fibrogenic activity of hydroxymetabolites of vitamin D3, while CYP27B1 is not required.

Vitamin D status alters genes involved in ovarian steroidogenesis in muskrat granulosa cells.

This study aims to explore the relationship between altered vitamin D (VitD3) status and ovarian steroidogenesis in muskrats during the breeding and non-breeding seasons. During the breeding season, the ovaries of muskrats were observably enlarged and increased in weight, accompanied by elevated serum and ovarian VitD3 status. Vitamin D receptor (VDR), VitD3 metabolic molecules (CYP2R1, CYP27B1, and CYP24A1), and steroidogenic enzymes were immunolocalized in the ovarian cells of muskrats. The mRNA levels of VDR, CYP2R1, CYP27B1, and steroidogenic enzymes were considerably higher during the breeding season compared to the non-breeding season. RNA-seq analysis revealed a prominent enrichment of vitamin-related and ovarian steroidogenesis pathways. Furthermore, the addition of 1,25(OH)2D3 to the muskrat granulosa cells in vitro increased VDR and steroidogenic enzymes mRNA levels and enhanced the 17ß-estradiol level. Overall, these findings supported that VitD3 promotes the secretion of steroid hormones, thereby affecting seasonal changes in ovarian function in the muskrats.

A PAI-1 antagonist ameliorates hypophosphatemia in the Hyp vitamin D-resistant rickets model mouse.

Congenital fibroblast growth factor 23 (FGF23)-related hypophosphatemic rickets/osteomalacia is a rare bone metabolism disorder characterized by hypophosphatemia and caused by genetic abnormalities that result in excessive secretion of FGF23. Hyp mice are a model of X-linked hypophosphatemia (XLH) caused by deletion of the PHEX gene and excessive production of FGF23. The purpose of this study was to investigate the potential of TM5614 as a therapeutic agent for the treatment of congenital FGF23-related hypophosphatemic rickets and osteomalacia in humans by administering TM5614 to Hyp mice and examining its curative effect on hypophosphatemia. After a single oral administration of TM5614 10 mg·kg-1 to female Hyp mice starting at 17 weeks of age, the serum phosphate concentration increased with a peak at 6 h after administration. ELISA confirmed that TM5614 administration decreased the intact FGF23 concentration in the blood. Expression of 25-hydroxyvitamin D-1α-hydroxylase protein encoded by Cyp27b1 mRNA in the kidney was suppressed in Hyp mice, and treatment with 10 mg·kg-1 of TM5614 normalized the expression of 25-hydroxyvitamin D-1α-hydroxylase protein and Cyp27b1 mRNA in the kidneys of these mice. Our data indicate that oral administration of TM5614 ameliorates hypophosphatemia in Hyp mice, suggesting that TM5614 may be an effective treatment for congenital FGF23-related hypophosphatemic rickets and osteomalacia.

Vitamin D3 Receptors and Metabolic Enzymes in Hen Reproductive Tissues.

In recent years, vitamin D3 has been revealed as an important regulator of reproductive processes in humans and livestock; however, its role in the female reproductive system of poultry is poorly known. The aim of this study was to examine vitamin D3 receptor (VDR and PDIA3) and metabolic enzyme (1α-hydroxylase and 24-hydroxylase) mRNA transcript and protein abundances, and protein localization within the hen ovary, oviductal shell gland, pituitary, liver, and kidney. We demonstrated, for the first time, the patterns of the relative mRNA and protein abundances of examined molecules in the ovary, dependent on follicle development and the layer of follicle wall, as well as in other examined organs. Immunohistochemically, PDIA3, 1α-hydroxylase, and 24-hydroxylase are localized in follicular theca and granulosa layers, luminal epithelium and tubular glands of the shell gland, pituitary, liver, and kidney. These results indicate that reproductive tissues have both receptors, VDR, primarily involved in genomic action, and PDIA3, probably participating in the rapid, non-genomic effect of vitamin D3. The finding of 1α-hydroxylase and 24-hydroxylase expression indicates that the reproductive system of chickens has the potential for vitamin D3 synthesis and inactivation, and may suggest that locally produced vitamin D3 can be considered as a significant factor in the orchestration of ovarian and shell gland function in hens. These results provide a new insight into the potential mechanisms of vitamin D3 action and metabolism in the chicken ovary and oviduct.

Impaired Growth Plate Maturation in XLH Is due to Both Excess FGF23 and Decreased 1,25-Dihydroxyvitamin D Signaling.

X-linked hypophosphatemia (XLH) is the most common form of hereditary hypophosphatemic rickets. The genetic basis for XLH is loss of function mutations in the phosphate-regulating endopeptidase X-linked (PHEX), which leads to increased circulating fibroblast growth factor 23 (FGF23). This increase in FGF23 impairs activation of vitamin D and attenuates renal phosphate reabsorption, leading to rickets. Previous studies have demonstrated that ablating FGF23 in the Hyp mouse model of XLH leads to hyperphosphatemia, high levels of 1,25-dihydroxyvitamin D, and is not associated with the development of rickets. Studies were undertaken to define a role for the increase in 1,25-dihydroxyvitamin D levels in the prevention of rickets in Hyp mice lacking FGF23. These mice were mated to mice lacking Cyp27b1, the enzyme responsible for activating vitamin D metabolites, to generate Hyp mice lacking both FGF23 and 1,25-dihydroxyvitamin D (FCH mice). Mice were fed a special diet to maintain normal mineral ion homeostasis. Despite normal mineral ions, Hyp mice lacking both FGF23 and Cyp27b1 developed rickets, characterized by an interrupted, expanded hypertrophic chondrocyte layer and impaired hypertrophic chondrocyte apoptosis. This phenotype was prevented when mice were treated with 1,25-dihydroxyvitamin D from day 2 until sacrifice on day 30. Interestingly, mice lacking FGF23 and Cyp27b1 without the PHEX mutation did not exhibit rickets. These findings define an essential PHEX-dependent, FGF23-independent role for 1,25-dihydroxyvitamin D in XLH and have important therapeutic implications for the treatment of this genetic disorder.

Association of DNA methylation of vitamin D metabolic pathway related genes with colorectal cancer risk.

BACKGROUND: Vitamin D might have anti-tumor effect, which is affected by the genes related to vitamin D metabolic pathway. Epigenetic mechanism may affect the expression level of vitamin D metabolic pathway related genes, then plays an important role in the occurrence and development of colorectal cancer. To date, no study has reported on the association between blood-based DNA methylation level of vitamin D metabolic pathway related genes and colorectal cancer risk. METHODS: A case-control study was conducted including 102 colorectal cancer cases and 102 sex- and age-frequency-matched controls in Guangzhou, China. CpG islands in the VDR, CYP24A1, CYP27B1 and CYP2R1 genes were chosen for DNA methylation analysis by MethylTarget sequencing. The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of DNA methylation levels for colorectal cancer. Taking the point with the largest Youden index as the boundary value, the cumulative methylation levels of vitamin D metabolic pathway related genes were divided into hypomethylation and hypermethylation. Unconditional multivariable logistical regression model was used to calculate the adjusted odds ratio (aOR) and 95% confidence intervals (95% CIs) after adjusting for potential confounders. RESULTS: Among 153 CpG sites, 8 CpG sites were significantly different between the cases and the controls. The cumulative methylation level of all CpG sites in CYP2R1 was inversely associated with the risk of colorectal cancer (aOR, 0.49; 95% CI, 0.26-0.91). However, no significant association was found between cumulative methylation levels of all CpG sites in VDR, CYP24A1 and CYP27B1 and colorectal cancer risk. Significant inverse association was observed between cumulative methylation level of significant CpG sites in VDR (aOR, 0.28; 95% CI, 0.16-0.51) and CYP24A1 (aOR, 0.19; 95% CI, 0.09-0.40) and colorectal cancer risk. There were no significant associations between cumulative methylation levels of significant CpG sites in CYP2R1 and CYP27B1 and colorectal cancer risk. CONCLUSIONS: This study indicated that the cumulative methylation levels of significant CpG sites in VDR and CYP24A1 and all CpG sites in CYP2R1 were inversely associated with colorectal cancer risk.

Seasonal changes of vitamin D3 and ovarian steroidogenesis in the wild ground squirrels (Citellus dauricus Brandt).

There is mounting evidence that vitamin D3 regulates female reproductive function critically, while little is known about the function of seasonally variable vitamin D3 in regulating ovarian steroidogenesis. This study examined the seasonal expressions of vitamin D receptor (VDR), vitamin D metabolic molecules (CYP2R1, CYP27B1, and CYP24A1), and steroidogenic enzymes (P450scc, 3ß-HSD, P450c17, and P450arom) in the ovaries of the wild ground squirrels (Citellus dauricus Brandt) during the different breeding seasons. VDR, CYP2R1, CYP27B1, and CYP24A1 were shown to be localized in different types of ovarian cells in the wild ground squirrels during the breeding and non-breeding seasons. Meanwhile, the mRNA levels of VDR, CYP2R1, CYP27B1, CYP11A1, HSD3B1, CYP17A1, and CYP19A1 in the ovaries were remarkably higher in the breeding season. Furthermore, RNA-seq data of ovaries revealed that 6036 genes were differentially expressed genes (DEGs); further analysis revealed that several DEGs known to be involved in ovarian steroidogenesis pathway and cellular response to vitamin D pathway were identified. In addition, during the breeding season, the concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone, and 17ß-estradiol were greater in the serum of the wild female ground squirrels. This observation was positively correlated with seasonal changes in the concentration of 25(OH)D3, supporting the fact that the 25(OH)D3 content in the ovaries was significantly higher in the breeding season. These findings suggested that seasonal changes in vitamin D3 might regulate the ovarian steroidogenesis of the wild female ground squirrels.

Molecular insights into mineralotropic hormone inter-regulation.

The regulation of mineral homeostasis involves the three mineralotropic hormones PTH, FGF23 and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Early research efforts focused on PTH and 1,25(OH)2D3 and more recently on FGF23 have revealed that each of these hormones regulates the expression of the other two. Despite early suggestions of transcriptional processes, it has been only recently that research effort have begun to delineate the genomic mechanisms underpinning this regulation for 1,25(OH)2D3 and FGF23; the regulation of PTH by 1,25(OH)2D3, however, remains obscure. We review here our molecular understanding of how PTH induces Cyp27b1 expression, the gene encoding the enzyme responsible for the synthesis of 1,25(OH)2D3. FGF23 and 1,25(OH)2D3, on the other hand, function by suppressing production of 1,25(OH)2D3. PTH stimulates the PKA-induced recruitment of CREB and its coactivator CBP at CREB occupied sites within the kidney-specific regulatory regions of Cyp27b1. PKA activation also promotes the nuclear translocation of SIK bound coactivators such as CRTC2, where it similarly interacts with CREB occupied Cyp27b1 sites. The negative actions of both FGF23 and 1,25(OH)2D3 appear to suppress Cyp27b1 expression by opposing the recruitment of CREB coactivators at this gene. Reciprocal gene actions are seen at Cyp24a1, the gene encoding the enzyme that degrades 1,25(OH)2D3, thereby contributing to the overall regulation of blood levels of 1,25(OH)2D3. Relative to PTH regulation, we summarize what is known of how 1,25(OH)2D3 regulates PTH suppression. These studies suggest that it is not 1,25(OH)2D3 that controls PTH levels in healthy subjects, but rather calcium itself. Finally, we describe current progress using an in vivo approach that furthers our understanding of the regulation of Fgf23 expression by PTH and 1,25(OH)2D3 and provide the first evidence that P may act to induce Fgf23 expression via a complex transcriptional mechanism in bone. It is clear, however, that additional advances will need to be made to further our understanding of the inter-regulation of each of these hormonal genes.

Impaired 1,25-dihydroxyvitamin D3 action underlies enthesopathy development in the Hyp mouse model of X-linked hypophosphatemia.

X-linked hypophosphatemia (XLH) is characterized by high serum fibroblast growth factor 23 (FGF23) levels, resulting in impaired 1,25-dihydroxyvitamin D3 (1,25D) production. Adults with XLH develop a painful mineralization of the tendon-bone attachment site (enthesis), called enthesopathy. Treatment of mice with XLH (Hyp) with 1,25D or an anti-FGF23 Ab, both of which increase 1,25D signaling, prevents enthesopathy. Therefore, we undertook studies to determine a role for impaired 1,25D action in enthesopathy development. Entheses from mice lacking vitamin D 1α-hydroxylase (Cyp27b1) (C-/-) had a similar enthesopathy to Hyp mice, whereas deletion of Fgf23 in Hyp mice prevented enthesopathy, and deletion of both Cyp27b1 and Fgf23 in mice resulted in enthesopathy, demonstrating that the impaired 1,25D action due to high FGF23 levels underlies XLH enthesopathy development. Like Hyp mice, enthesopathy in C-/- mice was observed by P14 and was prevented, but not reversed, with 1,25D therapy. Deletion of the vitamin D receptor in scleraxis-expressing cells resulted in enthesopathy, indicating that 1,25D acted directly on enthesis cells to regulate enthesopathy development. These results show that 1,25D signaling was necessary for normal postnatal enthesis maturation and played a role in XLH enthesopathy development. Optimizing 1,25D replacement in pediatric patients with XLH is necessary to prevent enthesopathy.

Genetic variants in the vitamin D pathway and their association with vitamin D metabolite levels: Detailed studies of an inner-city pediatric population suggest a modest but significant effect in early childhood.

OBJECTIVES: In a large cohort of healthy infants and toddlers 6-36 months of age (n = 776), we have been exploring the potential role of genetic variation in predisposition to vitamin D insufficiency. The genes encoding the key cytochrome P450 hydroxylases (CYP2R1, CYP24A1, and CYP27B1) harbour recurrent mutations of uncertain effect. This study was undertaken to look for biochemically relevant associations of these variants with inter-individual differences in vitamin D metabolism in an at-risk pediatric population. METHODS: Genotyping for CYP2R1-CT (c.-1127 C>T, rs10741657), CYP24A1-AG (c.-686A>G, rs111622401), and CYP27B1-CA (c.-1261 C>A, rs10877012) mutations were performed using SNaPshot assay, followed by Sanger sequencing confirmation. Vitamin D metabolites and vitamin D binding protein (DBP) were measured by established methods. RESULTS: In a multivariate regression model, with corrections for co-variates, subjects with the homozygous CYP2R1-TT variant had significantly higher concentrations of 25(OH)D, free 25(OH)D, and 24,25(OH)2D levels. In subjects with the CYP24A1-AG mutation, concentrations of 25(OH)D were significantly higher. CONCLUSIONS: The CYP2R1-TT and CYP24A1-AG variants have measurable effects on the vitamin D pathway. It seems unlikely that they will be clinically relevant in isolation, but they may be members of the large pool of infrequent mutations contributing to different risks for the vitamin D deficiency phenotype.

Mutations in the CYP27B1 gene cause vitamin D dependent rickets in pugs.

Rickets is a disorder of bone development and can be the result of either dietary or genetic causes. Here, related pugs from 2 litters were included. Three pugs had clinical signs including, lameness, bone deformities, and dyspnea. One other pug was found dead. Radiographs of 2 affected pugs, 5 and 6 months old, showed generalized widening, and irregular margination of the physes of both the appendicular and the axial skeleton with generalized decrease in bone opacity and bulbous swelling of the costochondral junctions. Two pugs had low serum calcium and 1,25 (OH)2 D3 concentrations. Test results further indicated secondary hyperparathyroidism with adequate concentrations of 25-hydroxyvitamin D. Necropsy revealed tongue-like projections of cartilage extending into the metaphysis consistent with rickets, loss of metaphyseal mineralization and lung pathology. Vitamin D-dependent rickets was diagnosed. A truncating mutation in the 1α-hydroxylase gene (CYP27B1) was identified by genome sequence analysis of the pugs with VDDR type 1A. Vitamin D-dependent rickets type 1A can occur in young pugs, and if left untreated is a life-threatening condition. Early medical intervention can reverse clinical signs and should be instituted as soon as possible.

Isochromosome 12p Formation Regulates Vitamin D Metabolism in Testicular Cancer.

Isochromosome 12p (iChr12p) is typical in almost all invasive testicular cancers. Increased copy number of genes on 12p is associated with the development of a clinically manifest tumor; however, the causative genes have not yet been identified. Chromosome 12 harbors many genes involved in Vitamin D metabolism. RNAseq analysis of Vitamin D receptor (VDR) genes from the TCGA cohort revealed that clustering of VDR expression signatures could differentiate between pure seminomas and non-seminomatous germ cell tumors (NSGCT). Using TCGA mRNA expression of anabolic (CYP2R1, CYP27A1 and CYP27B1) and catabolic (CYP24A1) Vitamin D enzymes, positive (PTHLH, IFNG, and TNF) and negative (FGF23) feedback regulators could also clearly distinguish between pure seminomas and NSGCT. We hypothesize that the regulation of Vitamin D metabolism might be disturbed through iChr12p formation, influencing testicular carcinogenesis via increased FGF23 and PTHLH expression. While FGF23 represses CYP27B1 and activates catabolism of active hormone, increased PTHLH secretion can lead to hypercalcemia via inactivation of VDR. In conclusion, testicular cancer is associated with extensive modifications in intratesticular Vitamin D homeostasis. Further research is needed to clarify whether Vitamin D deficiency causes the formation of iChr12p and whether Vitamin D deficiency via iChr12p genomic aberration is involved in testicular carcinogenesis.

Vitamin D and calcium co-therapy mitigates pre-established cadmium nephropathy by regulating renal calcium homeostatic molecules and improving anti-oxidative and anti-inflammatory activities in rat.

BACKGROUND: Cadmium (Cd) is a major environmental pollutant and chronic toxicity could induce nephropathy by increasing renal oxidative stress and inflammation. Although vitamin D (VD) and calcium (Ca) prophylactic treatments attenuated Cd-induced cell injury, none of the prior studies measure their renoprotective effects against pre-established Cd-nephropathy. AIMS: To measure the alleviating effects of VD and/or Ca single and dual therapies against pre-established nephrotoxicity induced by chronic Cd toxicity prior to treatment initiation. METHODS: Forty male adult rats were allocated into: negative controls (NC), positive controls (PC), Ca, VD and VC groups. The study lasted for eight weeks and all animals, except the NC, received CdCl2 in drinking water (44 mg/L) throughout the study. Ca (100 mg/kg) and/or VD (350 IU/kg) were given (five times/week) during the last four weeks to the designated groups. Subsequently, the expression of transforming growth factor-ß (TGF-ß1), inducible nitric oxide synthase (iNOS), neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), VD synthesising (Cyp27b1) and catabolizing (Cyp24a1) enzymes with VD receptor (VDR) and binding protein (VDBP) was measured in renal tissues. Similarly, renal expression of Ca voltage-dependent channels (CaV1.1/CaV3.1), store-operated channels (RyR1/ITPR1), and binding proteins (CAM/CAMKIIA/S100A1/S100B) were measured. Serum markers of renal function alongside several markers of oxidative stress (MDA/H2O2/GSH/GPx/CAT) and inflammation (IL-6/TNF-α/IL-10) together with renal cell apoptosis and expression of caspase-3 were also measured. RESULTS: The PC group exhibited hypovitaminosis D, hypocalcaemia, hypercalciuria, proteinuria, reduced creatinine clearance, and increased renal apoptosis/necrosis with higher caspase-3 expression. Markers of renal tissue damage (TGF-ß1/iNOS/NGAL/KIM-1), oxidative stress (MDA/H2O2), and inflammation (TNF-α/IL-1ß/IL-6) increased, whilst the antioxidants (GSH/GPx/CAT) and IL-10 decreased, in the PC group. The PC renal tissues also showed abnormal expression of Cyp27b1, Cyp24a1, VDR, and VDBP, alongside Ca-membranous (CaV1.1/CaV3.1) and store-operated channels (RyR1/ITPR1) and cytosolic Ca-binding proteins (CAM/CAMKIIA/S100A1/S100B). Although VD was superior to Ca monotherapy, their combination revealed the best mitigation effects by attenuating serum and renal tissue Cd concentrations, inflammation and oxidative stress, alongside modulating the expression of VD/Ca-molecules. CONCLUSIONS: This study is the first to show improved alleviations against Cd-nephropathy by co-supplementing VD and Ca, possibly by better regulation of Ca-dependent anti-oxidative and anti-inflammatory actions.

Local production of active vitamin D3 metabolites in breast cancer cells by CYP24A1 and CYP27B1.

The role of vitamin D3 and its metabolites in cancer and especially as a treatment option has been widely disputed. Clinicians noting low serum 25-hydroxyvitamin D3 [25(OH)D3] levels in their patients, recommend vitamin D3 supplementation as a method of reducing the risk of cancer; however, data supporting this are inconsistent. These studies rely on systemic 25(OH)D3 as an indicator of hormone status, but 25(OH)D3 is further metabolized in the kidney and other tissues under regulation by several factors. This study examined if breast cancer cells also possess the ability to metabolize 25(OH)D3, and if so, whether the resulting metabolites are secreted locally; if this ability reflects ERα66 status; and if they possess vitamin D receptors (VDR). To address this question, estrogen receptor alpha (ERα) positive (MCF-7) and ERα negative (HCC38 and MDA-MB-231) breast cancer cell lines were examined for expression of ERα66, ERα36, CYP24A1, CYP27B1, and VDR as well as for local production of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] after treatment with 25(OH)D3. The results showed that independent of ER status, breast cancer cells express the enzymes CYP24A1 and CYP27B1, which are responsible for converting 25(OH)D3 into its dihydroxylated forms. Moreover, these metabolites are produced at levels comparable to the levels observed in blood. They are positive for VDR, indicating that they can respond to 1α,25(OH)2D3, which can upregulate CYP24A1. These findings suggest that vitamin D metabolites may contribute to the tumorigenicity of breast cancer via autocrine and/or paracrine mechanisms.

Vitamin D metabolic pathway genes polymorphisms and vitamin D levels in association with neonatal hyperbilirubinemia in China: a single-center retrospective cohort study.

BACKGROUND: Neonatal hyperbilirubinemia (NH) is a major cause of hospitalization after birth. Previous studies indicated that vitamin D deficiency might play an important role in NH susceptibility, but the results were controversial. Meanwhile, there has been limited description of the association between vitamin D related genes single nucleotide polymorphisms (SNP) and NH susceptibility. We aimed to investigate the vitamin D metabolic pathway genes polymorphisms and vitamin D levels with NH susceptibility. METHODS: We retrospectively analyzed the clinical data, vitamin D levels and its metabolic pathway gene polymorphisms of 187 NH neonates and 149 controls at Tianjin Children's Hospital/Tianjin University Children's Hospital between April 2019 and August 2022. Vitamin D levels were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, and the genetic polymorphism of NADSYN1/DHCR7, GC, CYP2R1, CYP24A1 and CYP27B1 was detected by high resolution melting (HRM) analysis. RESULTS: The frequency of vitamin D deficiency (25(OH)D < 15 ng/mL) was significantly increased in the NH group compared to controls. TT genotype of rs12785878 and GT genotype of rs10877012 were protective factors of vitamin D deficiency and NH, and GT genotype and dominant model carriers of rs12785878 had a higher risk of severe NH than the GG genotype carriers (GT genotype: OR: 2.43; 95% CI: 1.22-4.86; P = 0.012, dominant model: OR: 1.97; 95% CI: 1.04-3.73; P = 0.037). GC gene haplotype was associated with vitamin D deficiency. No significant SNP-SNP and SNP-vitamin D levels interaction combinations were found. CONCLUSIONS: There were associations among NH, vitamin D deficiency and NADSYN1/DHCR7 and CYP27B1 polymorphisms, TT genotype of rs12785878 and GT genotype of rs10877012 could reduce the risk of vitamin D deficiency and NH. Furthermore, rs12785878 was significantly associated with severe NH.

Expression of Rat Cyp27b1 in HepG2 Cells Using Adenovirus Vector and Its Application to Evaluation of Self-Made and Commercially Available Anti-Cyp27b1 Antibodies.

Rat Cyp27b1 was successfully expressed in HepG2 cells using an adenovirus vector. High vitamin D 1α-hydroxylation activity was detected in them, whereas no activity was observed in non-infected cells. Similarly, vitamin D 1α-hydroxylation activity was also observed in HepG2 cells expressing Cyp27b1-Flag, which is tagged with a Flag at the C-terminus of Cyp27b1. Western blot analysis using an anti-Flag antibody showed a clear band of Cyp27b1-Flag. Next, we screened three types of anti-Cyp27b1 antibodies, which consist of two commercially available antibodies and our self-made antibody using Cyp27b1- or Cyp27b1-Flag expressing HepG2 cell lysate as a positive control. Surprisingly, Western blot analysis revealed that two commercially available antibodies did not detect Cyp27b1 but specifically detect other proteins. In contrast, our self-made antisera specifically detected Cyp27b1 and Cyp27b1-Flag in the HepG2 cells expressing Cyp27b1 or Cyp27b1-Flag. These commercially available antibodies have been used for the detection of Cyp27b1 by Western blotting and immunohistochemistry. Our results suggest that those data should be reanalyzed.

Polymorphisms in VDR, CYP27B1, CYP2R1, GC and CYP24A1 Genes as Biomarkers of Survival in Non-Small Cell Lung Cancer: A Systematic Review.

The objective of this systematic review was to provide a compilation of all the literature available on the association between single-nucleotide polymorphisms (SNPs) in the genes involved in the metabolic pathway of vitamin D and overall survival (OS) and progression-free survival (PFS) in patients with non-small cell lung cancer (NSCLC). This systematic review was conducted in accordance with the PRISMA guidelines. It included all the literature published up to 1 November 2022 and was carried out in four databases (Medline [PubMed], Scopus, Web of Science, and Embase), using the PICO strategy, with relevant keywords related to the objective. The quality of the studies included was evaluated with an assessment tool derived from the Strengthening the Reporting of Genetic Association Studies (STREGA) statement. Six studies were included in this systematic review. Our findings showed that the BsmI (rs1544410), Cdx-2 (rs11568820), FokI (rs2228570), ApaI (rs7975232), TaqI (rs731236), rs4646536, rs6068816, rs7041, and rs10741657 SNPs in the genes that play a part in vitamin D synthesis (CYP2R1, CYP27B1), transport (GC), and metabolism (CYP24A1), as well as in the vitamin D receptor (VDR), are associated with OS and/or PFS in patients with NSCLC. The SNPs in VDR have been the most extensively analyzed. This systematic review summed up the available evidence concerning the association between 13 SNPs in the main genes involved in the vitamin D metabolic pathway and prognosis in NSCLC. It revealed that SNPs in the VDR, CYP27B1, CYP24A1, GC, and CYP2R1 genes could have an impact on survival in this disease. These findings suggest the identification of prognostic biomarkers in NSCLC patients. However, evidence remains sparse for each of the polymorphisms examined, so these findings should be treated with caution.

Low vitamin D status is associated with inflammatory response in older patients with cerebral small vessel disease.

OBJECTIVES: This study aimed to determine the association of the NF-κB inflammatory signaling pathway with vitamin D status in older cerebral small vessel disease (SVD) patients. METHODS: We measured serum 25(OH)D, pro-and anti-inflammatory cytokines, and mRNA levels of the vitamin D-activating enzyme, CYP27B1, as well as NF-kB, COX-2, the chemokine-CCL2, IL-1ß, IL-6, TNF-α, TGF-ß, and IL-10, in cerebral SVD patients aged ≥60 years presenting with vascular dementia and age and gender-matched healthy controls. RESULTS: Low vitamin D status (insufficiency: serum 25(OH)D 12-20 ng/ml; deficiency: ≤12 ng/ml) was more prevalent among patients compared to controls. The mRNA levels of NF-kB, COX-2, CCL2, IL-1ß, and IL-6, and serum levels of pro-inflammatory cytokines (IL-1α, IL-1ß, IL-6, and TNF-α) were significantly higher in cases compared to controls. There was a significant correlation between CYP27B1 and NF-kB, COX-2, CCL2, and IL-1ß gene expression. Serum IL-1α, IL-1ß, and IL-6 concentrations and the expression of CCL-2, NF-kB2, and NF-kB3 genes were higher in vitamin D-deficient subjects compared to vitamin D-sufficient subjects. There was a significant negative correlation between serum 25(OH)D and IL-1α, IL-6, and TNF-α, and a positive correlation between 25(OH)D and IL-10. CONCLUSION: Low vitamin D is associated with an inflammatory response via NF-kB signaling, which could play a role in the etio-pathogenesis of SVD. Further large-scale studies are required to validate our findings.