Biallelic and monoallelic pathogenic variants in CYP24A1 and SLC34A1 genes cause idiopathic infantile hypercalcemia.

OBJECTIVE: Idiopathic infantile hypercalcemia (IIH) is a rare disorder of PTH-independent hypercalcemia. CYP24A1 and SLC34A1 gene mutations cause two forms of hereditary IIH. In this study, the clinical manifestations and molecular aspects of six new Chinese patients were investigated. METHODS: The clinical manifestations and laboratory study of six patients with idiopathic infantile hypercalcemia were analyzed retrospectively. RESULTS: Five of the patients were diagnosed with hypercalcemia, hypercalciuria, and bilateral medullary nephrocalcinosis. Their clinical symptoms and biochemical abnormalities improved after treatment. One patient presented at age 11 years old with arterial hypertension, hypercalciuria and nephrocalcinosis, but normal serum calcium. Gene analysis showed that two patients had compound heterozygous mutations of CYP24A1, one patient had a monoallelic CYP24A1 variant, and three patients had a monoallelic SLC34A1 variant. Four novel CYP24A1 variants (c.116G > C, c.287T > A, c.476G > A and c.1349T > C) and three novel SLC34A1 variants (c.1322 A > G, c.1697_1698insT and c.1726T > C) were found in these patients. CONCLUSIONS: A monoallelic variant of CYP24A1 or SLC34A1 gene contributes to symptomatic hypercalcemia, hypercalciuria and nephrocalcinosis. Manifestations of IIH vary with onset age. Hypercalcemia may not necessarily present after infancy and IIH should be considered in patients with nephrolithiasis either in older children or adults.

MicroRNA-125b regulates vitamin D resistance by targeting CYP24A1 in the progression of gestational diabetes mellitus.

Vitamin D deficiency is prevalent in pregnancy and has been associated with increased occurrences of preeclampsia, cesarean delivery, neonatal bacterial vaginosis, and gestational diabetes. CYP24A1, recognized as a key factor in vitamin D metabolism homeostasis, encodes 24-hydroxylase responsible for converting 25(OH)D3 and 1,25(OH)2D3 into inactive metabolites. Recently, we have reported CYP24A1 overexpression in patients with gestational diabetes mellitus (GDM) and trophoblast cells exposed to hyperglycemia. In this study, we explored miRNA-mediated regulation of CYP24A1 in GDM progression, validating our findings through silencing experiments in a trophoblast cell line. In silico tools identified miR-125b-5p as a putative target of CYP24A1. Expression analysis revealed downregulation of miR-125b-5p in blood samples from early GDM and GDM compared to healthy pregnant women, positively correlating with vitamin D levels. Hyperglycemic exposure in human trophoblastic cell lines (BeWo) decreased miR-125b-5p expression, concomitant with an increase in CYP24A1. To confirm the regulatory role of miR-125b on CYP24A1, we transfected BeWo cells with antimiR-125b or miR-125b mimic. AntimiR-125b transfection heightened CYP24A1 levels, while miR-125b mimic overexpression resulted in decreased CYP24A1 expression. These findings establish miR-125b as a regulator of CYP24A1. To explore the influence of miR-125b on vitamin D metabolism, trophoblast cells overexpressing miR-125b were treated with 0.1 and 1 µM calcitriol. Hyperglycemic conditions exhibited a reduction in CYP24A1 levels. Collectively, our results indicate that miR-125b may regulate vitamin D metabolism by targeting CYP24A1, contributing to GDM progression. These findings may pave the way for understanding vitamin D resistance in concurrent GDM development and identifying novel miRNAs targeting CYP24A1.

Dual effect of vitamin D3 on breast cancer-associated fibroblasts.

BACKGROUND: Cancer-associated fibroblasts (CAFs) play an important role in the tumor microenvironment. Despite the well-known in vitro antitumoral effect of vitamin D3 (VD3), its impact on breast CAFs is almost unknown. In this study, we analyzed the ex vivo effects of calcitriol on CAFs isolated from breast cancer tissues. METHODS: CAFs were cultured with 1 and 10 nM calcitriol and their phenotype; gene expression, protein expression, and secretion were assessed. Calcitriol-treated CAFs-conditioned media (CM) were used to analyze the effect of CAFs on the migration and protein expression of MCF-7 and MDA-MB-231 cells. RESULTS: Tumor tissues from VD3-deficient patients exhibited lower levels of ß-catenin and TGFß1, along with higher levels of CYP24A1 compared to VD3-normal patients. In VD3-deficient patients, CAF infiltration was inversely associated with CYP24A1 levels and positively correlated with OPN levels. Calcitriol diminished CAFs' viability, but this effect was weaker in premenopausal and VD3-normal patients. Calcitriol reduced mRNA expression of CCL2, MMP9, TNC, and increased PDPN, SPP1, and TIMP1. It also decreased the secretion of CCL2, TNC, and the activity of MMP-2, while increasing cellular levels of TIMP1 in CAFs from all patient groups. In nonmetastatic and postmenopausal patients, PDPN surface expression increased, and CAFs CM from these groups decreased MCF-7 cell migration after ex vivo calcitriol treatment. In premenopausal and VD3-deficient patients, calcitriol reduced IDO1 expression in CAFs. Calcitriol-treated CAFs CM from these patients decreased OPN expression in MCF-7 and/or MDA-MB-231 cells. However, in premenopausal patients, calcitriol-treated CAFs CM also decreased E-cadherin expression in both cell lines. CONCLUSION: The effects of calcitriol on breast CAFs, both at the gene and protein levels, are complex, reflecting the immunosuppressive or procancer properties of CAFs. The anticancer polarization of CAFs following ex vivo calcitriol treatment may result from decreased CCL2, TNC (gene and protein), MMP9, and MMP-2, while the opposite effect may result from increased PDPN, TIMP1 (gene and protein), and SPP1. Despite these multifaceted effects of calcitriol on molecule expression, CAFs' CMs from nonmetastatic and postmenopausal patients treated ex vivo with calcitriol decreased the migration of MCF-7 cells.

Short-term fasting of mice elevates circulating fibroblast growth factor 23 (FGF23).

AIMS: Phosphate and vitamin D homeostasis are controlled by fibroblast growth factor 23 (FGF23) from bone suppressing renal phosphate transport and enhancing 24-hydroxylase (Cyp24a1), thereby inactivating 1,25(OH)2 D3 . Serum FGF23 is correlated with outcomes in several diseases. Fasting stimulates the production of ketone bodies. We hypothesized that fasting can induce FGF23 synthesis through the production of ketone bodies. METHODS: UMR106 cells and isolated neonatal rat ventricular myocytes (NRVM) were treated with ketone body ß-hydroxybutyrate. Mice were fasted overnight, fed ad libitum, or treated with ß-hydroxybutyrate. Proteins and further blood parameters were determined by enzyme-linked immunoassay (ELISA), western blotting, immunohistochemistry, fluorometric or colorimetric methods, and gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: ß-Hydroxybutyrate stimulated FGF23 production in UMR106 cells in a nuclear factor kappa-light-chain enhancer of activated B-cells (NFκB)-dependent manner, and in NRVMs. Compared to fed animals, fasted mice exhibited higher ß-hydroxybutyrate and FGF23 serum levels (based on assays either detecting C-terminal or intact, biologically active FGF23 only), cardiac, pancreatic, and thymic Fgf23 and renal Cyp24a1 expression, and lower 1,25(OH)2 D3 serum concentration as well as renal Slc34a1 and αKlotho (Kl) expression. In contrast, Fgf23 expression in bone and serum phosphate, calcium, plasma parathyroid hormone (PTH) concentration, and renal Cyp27b1 expression were not significantly affected by fasting. CONCLUSION: Short-term fasting increased FGF23 production, as did administration of ß-hydroxybutyrate, effects possibly of clinical relevance in view of the increasing use of FGF23 as a surrogate parameter in clinical monitoring of diseases. The fasting state of patients might therefore affect FGF23 tests.

Interactions of human mitochondrial Ferredoxin 1 (Adrenodoxin) by NMR; modulation by cytochrome P450 substrate and by truncation of the C-terminal tail.

Human Ferredoxin 1, also referred to as Adrenodoxin (Adx), is the sole electron carrier supporting the function of all seven mitochondrial cytochrome P450 (CYP) enzymes. Adx utilizes conserved negatively charged residues along its α-helix3 to interact with either the proximal surface of CYP enzymes or the binding surface of Adrendodoxin Reductase (AdR). However, in the oxidized state, Adx assumes a monomer-homodimer equilibrium that requires the presence of its unstructured C-terminal tail. Crystallographic structures of full-length human Adx dimers indicate that part of the binding surface necessary for its interactions with CYPs or with AdR is partially occluded by the dimer interface. In this study, protein NMR spectroscopy was used to interrogate the interactions between full-length (2-124) or truncated monomeric (2-108) human Adx and human CYP24A1 (with and without its vitamin-D substrate) as well as interactions with AdR. Here, monomeric Adx induced a similar pattern of peak broadening as that induced by addition of CYP24A1 substrate, consistent with a 1:1 Adx:CYP interaction as the functional complex. Additionally, removal of the C-terminal tail appears to enhance the interaction with AdR, despite removal of some of the AdR contacts in the tail region. This finding was also supported by an NMR competition assay. These findings suggest that the Adx dimers do not undergo meaningful interactions with either CYP or AdR, but may instead be responsible for regulating access to monomeric Adx. These conclusions are discussed in the context of a revised model of the Adx electron shuttle mechanism.

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.

Differential Metabolic Stability of 4α,25- and 4ß,25-Dihydroxyvitamin D3 and Identification of Their Metabolites.

Vitamin D3 (1) is metabolized by various cytochrome P450 (CYP) enzymes, resulting in the formation of diverse metabolites. Among them, 4α,25-dihydroxyvitamin D3 (6a) and 4ß,25-dihydroxyvitamin D3 (6b) are both produced from 25-hydroxyvitamin D3 (2) by CYP3A4. However, 6b is detectable in serum, whereas 6a is not. We hypothesized that the reason for this is a difference in the susceptibility of 6a and 6b to CYP24A1-mediated metabolism. Here, we synthesized 6a and 6b, and confirmed that 6b has greater metabolic stability than 6a. We also identified 4α,24R,25- and 4ß,24R,25-trihydroxyvitamin D3 (16a and 16b) as metabolites of 6a and 6b, respectively, by HPLC comparison with synthesized authentic samples. Docking studies suggest that the ß-hydroxy group at C4 contributes to the greater metabolic stability of 6b by blocking a crucial hydrogen-bonding interaction between the C25 hydroxy group and Leu325 of CYP24A1.

USP11-mediated LSH deubiquitination inhibits ferroptosis in colorectal cancer through epigenetic activation of CYP24A1.

Ferroptosis is an iron-dependent form of regulated cell death characterized by lipid peroxidation. Colorectal cancer (CRC) cells evade ferroptosis despite their requirement of substantial iron and reactive oxygen species (ROS) to sustain active metabolism and extensive proliferation. However, the underlying mechanism is unclear. Herein, we report the role of lymphoid-specific helicase (LSH), a chromatin-remodeling protein, in suppressing erastin-induced ferroptosis in CRC cells. We demonstrate that erastin treatment leads to dose- and time-dependent downregulation of LSH in CRC cells, and depletion of LSH increases cell sensitivity to ferroptosis. Mechanistically, LSH interacts with and is stabilized by ubiquitin-specific protease 11 (USP11) via deubiquitination; this interaction was disrupted by erastin treatment, resulting in increased ubiquitination and LSH degradation. Moreover, we identified cytochrome P450 family 24 subfamily A member 1 (CYP24A1) as a transcriptional target of LSH. LSH binds to the CYP24A1 promoter, promoting nucleosome eviction and reducing H3K27me3 occupancy, thus leading to transcription of CYP24A1. This cascade inhibits excessive intracellular Ca2+ influx, thereby reducing lipid peroxidation and ultimately conferring resistance to ferroptosis. Importantly, aberrant expression of USP11, LSH, and CYP24A1 is observed in CRC tissues and correlates with poor patient prognosis. Taken together, our study demonstrates the crucial role of the USP11/LSH/CYP24A1 signaling axis in inhibiting ferroptosis in CRC, highlighting its potential as a therapeutic target in CRC treatment.

Synthesis of New 26,27-Difluoro- and 26,26,27,27-Tetrafluoro-25-hydroxyvitamin D3: Effects of Terminal Fluorine Atoms on Biological Activity and Half-life.

As an extension of our research on providing a chemical library of side-chain fluorinated vitamin D3 analogues, we newly designed and synthesized 26,27-difluoro-25-hydroxyvitamin D3 (1) and 26,26,27,27-tetrafluoro-25-hydroxyvitamin D3 (2) using a convergent method applying the Wittig-Horner coupling reaction between CD-ring ketones (13, 14) and A-ring phosphine oxide (5). The basic biological activities of analogues, 1, 2, and 26,26,26,27,27,27-hexafluoro-25-hydroxyvitamin D3 [HF-25(OH)D3] were examined. Although the tetrafluorinated new compound 2 exhibited higher binding affinity for vitamin D receptor (VDR) and resistance to CYP24A1-dependent metabolism compared with the difluorinated 1 and its non-fluorinated counterpart 25-hydroxyvitamin D3 [25(OH)D3], HF-25(OH)D3 showed the highest activity among these compounds. Osteocalcin promoter transactivation activity of these fluorinated analogues was tested, and it decreased in the order of HF-25(OH)D3, 2, 1, and 25(OH)D3 in which HF-25(OH)D3 showed 19-times greater activity than the natural 25(OH)D3.

Inactivation of vitamin D2 metabolites by human CYP24A1.

Vitamin D is found in two forms in humans, D3 produced in the skin and D2 solely from the diet. Both 25-hydroxyvitamin D (25(OH)D) and 1,25-dihydroxyvitamin D (1,25(OH)2D) are oxidised and inactivated by CYP24A1, a tightly regulated mitochondrial enzyme that controls serum levels of these secosteroids. The pathways of oxidation of 25(OH)D2 and 1,25(OH)2D2, particularly 25(OH)D2, by human CYP24A1 are not well characterized. The aim of this study was to further elucidate these pathways, and to compare the kinetics of metabolism of 25(OH)D2 and 1,25(OH)2D2 with their vitamin D3 counterparts. We used expressed and partially purified human CYP24A1 with substrates dissolved in the membrane of phospholipid vesicles, to mimic the inner mitochondrial membrane. We found that the major pathways for side chain oxidation of 25(OH)D2 and 1,25(OH)2D2 were identical and that predominant intermediates of 25(OH)D2 metabolism could be converted to the corresponding intermediates in the pathway of 1,25(OH)2D2 oxidation by 1α-hydroxylation by CYP27B1. The initial steps in the CYP24A1-mediated oxidation involved hydroxylation at the C24R position, and another unknown position where the alcohol was oxidised to an aldehyde. The 24R-hydroxylation was followed by hydroxylation at C26 or C28, or cleavage between C24 and C25 to produce the 24-oxo-25,26,27-trinor derivative. All of these products were further oxidised, with 24-oxo-25,26,27-trinor-1(OH)D2 giving a product tentatively identified as 24-oxo-25,26,27-trinor-1,28(OH)2D2. The catalytic efficiency (kcat/Km) of CYP24A1 for initial 25(OH)D2 hydroxylation was similar to that for 25(OH)D3, indicating that they have similar rates of inactivation at low substrate concentrations, supporting that vitamins D2 and D3 are equally effective in maintaining serum 25(OH)D concentrations. In contrast, the kcat/Km value for 1,25(OH)2D3 was almost double that for 1,25(OH)2D2 indicating a lower rate of inactivation of 1,25(OH)2D2 at a low substrate concentration, suggesting that it has increased metabolic stability in vivo.

Role of Coactivator Associated Arginine Methyltransferase 1 (CARM1) in the Regulation of the Biological Function of 1,25-Dihydroxyvitamin D3.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the hormonally active form of vitamin D, activates the nuclear vitamin D receptor (VDR) to mediate the transcription of target genes involved in calcium homeostasis as well as in non-classical 1,25(OH)2D3 actions. In this study, CARM1, an arginine methyltransferase, was found to mediate coactivator synergy in the presence of GRIP1 (a primary coactivator) and to cooperate with G9a, a lysine methyltransferase, in 1,25(OH)2D3 induced transcription of Cyp24a1 (the gene involved in the metabolic inactivation of 1,25(OH)2D3). In mouse proximal renal tubule (MPCT) cells and in mouse kidney, chromatin immunoprecipitation analysis demonstrated that dimethylation of histone H3 at arginine 17, which is mediated by CARM1, occurs at Cyp24a1 vitamin D response elements in a 1,25(OH)2D3 dependent manner. Treatment with TBBD, an inhibitor of CARM1, repressed 1,25(OH)2D3 induced Cyp24a1 expression in MPCT cells, further suggesting that CARM1 is a significant coactivator of 1,25(OH)2D3 induction of renal Cyp24a1 expression. CARM1 was found to act as a repressor of second messenger-mediated induction of the transcription of CYP27B1 (involved in the synthesis of 1,25(OH)2D3), supporting the role of CARM1 as a dual function coregulator. Our findings indicate a key role for CARM1 in the regulation of the biological function of 1,25(OH)2D3.

Transcriptome-wide profile of 1α,25 dihydroxyvitamin D3 in HTR-8/SVneo cells.

AIM: Vitamin D3 has been implicated in multiple reproductive events, whereas the effect of its bioactive metabolite 1α, 25 dihydroxyvitamin D3 (1,25(OH) 2 D3 ) on transcriptome profile of the placenta is unclear. The aim of this article is to determine transcriptome-wide profile caused by 1,25(OH) 2 D3 in human placental trophoblast cells. METHODS: We performed RNA sequencing after stimulation of HTR-8/SVneo cells with 0.1, 1, 10, and 100 nM 1,25(OH)2 D3 for 24 h, identified differentially expressed genes by edgeR package (version 3.38.4), and analyzed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways by webtool Metascape. Also, common genes and specific genes in different concentrations of 1,25(OH) 2 D3 were identified. RESULTS: There were 180, 158, 161, and 174 differentially expressed genes after 0.1, 1, 10, and 100 nM 1,25(OH) 2 D3 stimulation, respectively. KEGG pathway analysis displayed that "lipid and atherosclerosis" were significantly enriched at 0.1 and 1 nM 1,25(OH)2 D3 , while "cytokine-cytokine receptor interaction," "TGF-beta signaling pathway" and "hippo signaling pathway" were significantly enriched in 1, 10, and 100 nM 1,25(OH)2 D3 . CYP24A1 was a significantly expressed common gene. UCP3 was significantly expressed in low concentrations and might affect energy metabolism. TCF24, EIF3CL, ABCD2, EPHA7, CRLF1, and SECTM1 were specific genes at physiological concentration. Similarly, SPDYE1, IQUB, IL18R1, and ZNF713 were considered as specific genes at supraphysiological concentration. CONCLUSIONS: 1,25(OH)2 D3 mainly affected the expression of CYP24A1 gene in HTR-8/SVneo cells. Specific genes accounted for the majority of differentially expressed genes at different concentrations. However, their functions need to be further confirmed.

The CYP24A1 gene variant rs2762943 is associated with low serum 1,25-dihydroxyvitamin D levels in multiple sclerosis patients.

BACKGROUND AND PURPOSE: Vitamin D is considered to play a role in multiple sclerosis (MS) etiopathogenesis. A polymorphism in the CYP24A1 gene, rs2762943, was recently identified that was associated with an increased MS risk. CYP24A1 encodes a protein involved in the catabolism of the active form of vitamin D. The immunological effects of carrying the rs2762943 risk allele were investigated, as well as its role as genetic modifier. METHODS: Serum levels of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D (1,25(OH)2 D) were measured in a cohort of 167 MS patients. In a subgroup of patients, expression levels of major histocompatibility complex class II and co-stimulatory molecules were determined by flow cytometry, and serum levels of pro-inflammatory (interferon gamma, granulocyte macrophage colony-stimulating factor, C-X-C motif chemokine ligand 13) and anti-inflammatory (interleukin 10) cytokines and neurofilament light chain were measured by single-molecule array assays. The effect of the rs2762943 polymorphism on disease activity and disability measures was evaluated in 340 MS patients. RESULTS: Compared to non-carriers, carriers of the rs2762943 risk allele were characterized by reduced levels of 1,25(OH)2 D (p = 0.0001) and elevated levels of interferon gamma (p = 0.03) and granulocyte macrophage colony-stimulating factor (p = 0.008), whereas no significant differences were observed for the other markers. The presence of the rs2762943 risk allele had no significant impact on disease activity and disability outcomes during follow-up. However, risk allele carriers were younger at disease onset (p = 0.04). CONCLUSIONS: These findings suggest that the CYP24A1 rs2762943 polymorphism plays a more important role in MS susceptibility than in disease prognosis and is associated with lower 1,25(OH)2 D levels and a heightened pro-inflammatory environment in MS patients.

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.

Low Magnesium Concentration Enforces Bone Calcium Deposition Irrespective of 1,25-Dihydroxyvitamin D3 Concentration.

Efficient coordination between Mg2+ and vitamin D maintains adequate Ca2+ levels during lactation. This study explored the possible interaction between Mg2+ (0.3, 0.8, and 3 mM) and 1,25-dihydroxyvitamin D3 (1,25D; 0.05 and 5 nM) during osteogenesis using bovine mesenchymal stem cells. After 21 days, differentiated osteocytes were subjected to OsteoImage analysis, alkaline phosphatase (ALP) activity measurements, and immunocytochemistry of NT5E, ENG (endoglin), SP7 (osterix), SPP1 (osteopontin), and the BGLAP gene product osteocalcin. The mRNA expression of NT5E, THY1, ENG, SP7, BGLAP, CYP24A1, VDR, SLC41A1, SLC41A2, SLC41A3, TRPM6, TRPM7, and NIPA1 was also assessed. Reducing the Mg2+ concentration in the medium increased the accumulation of mineral hydroxyapatite and ALP activity. There was no change in the immunocytochemical localization of stem cell markers. Expression of CYP24A1 was higher in all groups receiving 5 nM 1,25D. There were tendencies for higher mRNA abundance of THY1, BGLAP, and NIPA1 in cells receiving 0.3 mM Mg2+ and 5 nM 1,25D. In conclusion, low levels of Mg2+ greatly enhanced the deposition of bone hydroxyapatite matrix. The effect of Mg2+ was not modulated by 1,25D, although the expression of certain genes (including BGLAP) tended to be increased by the combination of low Mg2+ and high 1,25D concentrations.

Suppression of the vitamin D metabolizing enzyme CYP24A1 provides increased sensitivity to chemotherapeutic drugs in breast cancer.

Vitamin D is an essential nutrient for the human body not only for the metabolism of calcium but also for homeostasis. Vitamin D contributes to cell fate decisions, including cell proliferation, differentiation and viability. Accumulated epidemiological data suggest a relationship between vitamin D deficiency and carcinogenesis in numerous organs. Furthermore, it is known that the expression of the vitamin D metabolizing enzyme, cytochrome P450 family 24 subtype A1 (CYP24A1), is increased in different types of human malignancy including breast carcinoma. However, the pathological relevance of elevated CYP24A1 expression level requires further clarification. In the present study, it was demonstrated that CYP24A1 promoted the oncogenic property of breast carcinoma cells. Consistent with previous reports, it was demonstrated that the expression of CYP24A1 was elevated in invasive breast carcinoma and significantly decreased the overall survival of patients with invasive breast carcinoma. Importantly, suppression of CYP24A1 expression significantly enhanced cell death sensitivity to two anticancer drugs with pharmacologically different modes of action, cisplatin and gefitinib. The results of the present study suggest the possibility of CYP24A1­inhibiting therapy as a novel therapy in breast cancer with overexpression of CYP24A1.

Rifampin monotherapy for children with idiopathic infantile hypercalcemia.

Idiopathic Infantile Hypercalcemia (IIH) is characterized by hypercalcemia and hypercalciuria owing to PTH-independent increases in circulating concentrations of 1,25(OH)2D. At least 3 forms of IHH can be distinguished genetically and mechanistically: infantile hypercalcemia-1 (Hypercalcemia, Infantile, 1; HCINF1) due to CYP24A1 mutations results in decreased inactivation of 1,25(OH)2D; HCINF2 due to SLC34A1 mutations results in excessive 1,25(OH)2D production; and HCINF3 in which a variety of gene variants of uncertain significance (VUS) have been identified and where the mechanism for increased 1,25 (OH)2D is unclear. Conventional management with dietary calcium and vitamin D restriction has only limited success. Induction of the P450 enzyme CYP3A4 by rifampin can provide an alternate pathway for inactivation of 1,25(OH)2D that is useful in HCINF1 and may be effective in other forms of IIH. We sought to assess the efficacy of rifampin to decrease levels of serum 1,25(OH)2D and calcium, and urinary calcium concentrations in subjects with HCINF3, and to compare the response to a control subject with HCINF1. Four subjects with HCINF3 and the control subject with HCINF1 completed the study using rifampin 5 mg/kg/day and 10 mg/kg/day each for 2 months separated by a 2-month washout period. Patients had age-appropriate intake of dietary calcium plus 200 IU vitamin D/day. Primary outcome was efficacy of rifampin to lower serum concentrations of 1,25(OH)2D. The secondary outcomes included the reduction of serum calcium, urinary calcium excretion (as random urine calcium: creatinine (ca:cr) ratio) and serum 1,25(OH)2D/PTH ratio. Rifampin was well tolerated and induced CYP3A4 at both doses in all subjects. The control subject with HCINF1 showed significant response to both rifampin doses with decreases in the serum concentration of 1,25(OH)2D and the 1,25(OH)2D/PTH ratio while the serum and urine ca:cr levels were unchanged. The four patients with HCINF3 showed reductions in 1,25(OH)2D and urinary ca:cr after 10 mg/kg/d, but hypercalcemia did not improve and there were variable responses in 1,25(OH)2D/PTH ratios. These results support further longer-term studies to clarify the usefulness of rifampin as a medical therapy for IIH.

Decreased Vitamin D Levels and Altered Placental Vitamin D Gene Expression at High Altitude: Role of Genetic Ancestry.

High-altitude hypoxia challenges reproduction; particularly in non-native populations. Although high-altitude residence is associated with vitamin D deficiency, the homeostasis and metabolism of vitamin D in natives and migrants remain unknown. We report that high altitude (3600 m residence) negatively impacted vitamin D levels, with the high-altitude Andeans having the lowest 25-OH-D levels and the high-altitude Europeans having the lowest 1α,25-(OH)2-D levels. There was a significant interaction of genetic ancestry with altitude in the ratio of 1α,25-(OH)2-D to 25-OH-D; with the ratio being significantly lower in Europeans compared to Andeans living at high altitude. Placental gene expression accounted for as much as 50% of circulating vitamin D levels, with CYP2R1 (25-hydroxylase), CYP27B1 (1α-hydroxylase), CYP24A1 (24-hydroxylase), and LRP2 (megalin) as the major determinants of vitamin D levels. High-altitude residents had a greater correlation between circulating vitamin D levels and placental gene expression than low-altitude residents. Placental 7-dehydrocholesterol reductase and vitamin D receptor were upregulated at high altitude in both genetic-ancestry groups, while megalin and 24-hydroxylase were upregulated only in Europeans. Given that vitamin D deficiency and decreased 1α,25-(OH)2-D to 25-OH-D ratios are associated with pregnancy complications, our data support a role for high-altitude-induced vitamin D dysregulation impacting reproductive outcomes, particularly in migrants.