CYP24A1 affected macrophage polarization through degradation of vitamin D as a candidate biomarker for ovarian cancer prognosis.

Ovarian cancer (OC) is a fatal gynecological malignancy with a poor prognosis in which mitochondria-related genes are involved deeply. In this study, we aim to screen mitochondria-related genes that play a role in OC prognosis and investigate its effects. Through single-cell sequencing technology and bioinformatics analysis, including TCGA ovarian cancer data analysis, gene expression signature analysis (GES), immune infiltration analysis, Gene Ontology (GO) enrichment analysis, Gene Set Enrichment Analysis (GSEA), and Principal Component Analysis (PCA), our findings revealed that CYP24A1 regulated macrophage polarization through vitamin D (VD) degradation and served as a target gene for the second malignant subtype of OC through bioinformatics analyses. For further validation, the expression and function of CYP24A1 in OC cells was investigated. And the expression of CYP24A1 was much higher in carcinoma than in paracancerous tissue, whereas the VD content decreased in the OC cell lines with CYP24A1 overexpression. Moreover, macrophages were polarized towards M1 after the intervention of VD-treated OC cell lines and inhibited the malignant phenotypes of OC. However, the effect could be reversed by overexpressing CYP24A1, resulting in the polarization of M2 macrophages, thereby promoting tumor progression, as verified by constructing xenograft models in vitro. In conclusion, our findings suggested that CYP24A1 induced M2 macrophage polarization through interaction with VD, thus promoting the malignant progression of OC.

Exploring the role of vitamin D-VDR pathway in pemphigus foliaceous: a novel perspective on disease pathogenesis.

Several auto-immune diseases have been linked to vitamin D deficiency as a contributing environmental factor. Its pleiotropic effects on the immune system, especially its essential role in maintaining immune tolerance, make the vitamin D pathway of great interest. In this study, we focused on Pemphigus foliaceous (PF) in Tunisian population. we aimed to quantify the Serum 25[OH]D levels using chemiluminescence assay and to analyze the differential expression of the VDR, CYP27B1 and CYP24A1 genes in the circulating blood cells and lesional skin tissue of PF patients using Q-PCR. A genetic explanation was then sought to explore any direct relationship between tag polymorphisms and the inherited features of PF. Results confirmed a vitamin D hypovitaminosis in Tunisian PF patients. Interestingly, a differential gene expression correlated to the disease stratification was noted. Indeed, at the systemic level, an upregulation of VDR and CYP27B1 genes was observed in healthy controls compared to PF patients. Notably, in lesional skin tissue, the clinical and serological remission phase was correlated with high transcriptional levels of the VDR gene and conversely a drop in expression of the CYP24A1 gene. Genetic analysis indicated the involvement of the most appealing polymorphisms, rs2228570 and poly (A) microsatellite, in PF etiopathogenesis. Indeed, CAC13 haplotype was associated with a higher risk of PF development. Our findings suggest that alterations in the vitamin D-VDR pathway may influence PF physiopathology, making this pathway a potential target for pharmacological modulation, especially for cortico-resistant PF patients.

Effects of High-Dose Vitamin D Supplementation on Placental Vitamin D Metabolism and Neonatal Vitamin D Status.

Vitamin D (vitD) deficiency (25-hydroxy-vitamin D < 50 nmol/L) is common in pregnancy and associated with an increased risk of adverse pregnancy outcomes. High-dose vitD supplementation is suggested to improve pregnancy health, but there is limited knowledge about the effects on placental vitD transport and metabolism and the vitD status of newborns. Comparing the current standard maternal supplementation, 10 µg/day to a 90 µg vitD supplement, we investigated placental gene expression, maternal vitD transport and neonatal vitD status. Biological material was obtained from pregnant women randomized to 10 µg or 90 µg vitD supplements from week 11-16 onwards. Possible associations between maternal exposure, neonatal vitD status and placental expression of the vitD receptor (VDR), the transporters (Cubilin, CUBN and Megalin, LRP2) and the vitD-activating and -degrading enzymes (CYP24A1, CYP27B1) were investigated. Maternal vitD-binding protein (VDBP) was determined before and after supplementation. Overall, 51% of neonates in the 10 µg vitD group were vitD-deficient in contrast to 11% in the 90 µg group. High-dose vitD supplementation did not significantly affect VDBP or placental gene expression. However, the descriptive analyses indicate that maternal obesity may lead to the differential expression of CUBN, CYP24A1 and CYP27B1 and a changed VDBP response. High-dose vitD improves neonatal vitD status without affecting placental vitD regulation.

Unraveling the role of the vitamin D-VDR pathway in pemphigus vulgaris from Tunisian patients.

Vitamin D dysregulation has been recognized as a factor that may cause or aggravate autoimmunity. Vitamin D deficiency was found to be common in pemphigus vulgaris (PV) in different populations. This study aimed to investigate the vitamin D-VDR pathway in PV in the Tunisian population. A serological study was carried out to determine the vitamin D status in newly diagnosed PV patients. CYP27B1, CYP24A1 and VDR mRNA expression was assessed using quantitative real-time PCR in peripheral blood mononuclear cells (PBMC) from untreated newly diagnosed and treated PV patients. In addition, a genetic study was accomplished on VDR polymorphisms to investigate the changes in VDR gene expression. Overall, the serological study confirmed the hypovitaminosis D in newly diagnosed PV patients. Vitamin D-VDR pathway gene expression showed downregulation of CYP27B1 and CYP24A1 mRNA in first-discovery patients compared to healthy controls, while VDR mRNA was highly expressed in newly diagnosed PV patients. Moreover, CYP27B1, CYP24A1 and VDR mRNA were significantly upregulated in chronic disease severity groups compared to mild disease groups. The genetic study showed low VDR gene expression in carriers of FokI > CC genotype, which was more frequent among PV patients, and FokI > C-TaqI > C-ApaI > A-polyA > A16 haplotype, suggesting that the VDR gene polymorphisms testing can provide useful information for PV treatment decision-making. In conclusion, our findings underline the impact of vitamin D-VDR pathway disruption in the PV pathophysiology in Tunisian patients.

Zika virus infection suppresses CYP24A1 and CAMP expression in human monocytes.

Monocytes are the primary targets of Zika virus (ZIKV) and are associated with ZIKV pathogenesis. Currently, there is no effective treatment for ZIKV infection. It is known that 1,25-dihydroxy vitamin D3 (VitD3) has strong antiviral activity in dengue virus-infected macrophages, but it is unknown whether VitD3 inhibits ZIKV infection in monocytes. We investigated the relationship between ZIKV infection and the expression of genes of the VitD3 pathway, as well as the inflammatory response of infected monocytes in vitro. ZIKV replication was evaluated using a plaque assay, and VitD3 pathway gene expression was analyzed by RT-qPCR. Pro-inflammatory cytokines/chemokines were quantified using ELISA. We found that VitD3 did not suppress ZIKV replication. The results showed a significant decrease in the expression of vitamin D3 receptor (VDR), cytochrome P450 family 24 subfamily A member 1 (CYP24A1), and cathelicidin antimicrobial peptide (CAMP) genes upon ZIKV infection. Treatment with VitD3 was unable to down-modulate production of pro-inflammatory cytokines, except TNF-α, and chemokines. This suggests that ZIKV infection inhibits the expression of VitD3 pathway genes, thereby preventing VitD3-dependent inhibition of viral replication and the inflammatory response. This is the first study to examine the effects of VitD3 in the context of ZIKV infection, and it has important implications for the role of VitD3 in the control of viral replication and inflammatory responses during monocyte infection.

2α-Substituted Vitamin D Derivatives Effectively Enhance the Osteoblast Differentiation of Dedifferentiated Fat Cells.

The active form of vitamin D3, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], is a principal regulator of calcium homeostasis through activation of the vitamin D receptor (VDR). Previous studies have shown that 2α-(3-hydroxypropyl)-1,25D3 (O1C3) and 2α-(3-hydroxypropoxy)-1,25D3 (O2C3), vitamin D derivatives resistant to inactivation enzymes, can activate VDR, induce leukemic cell differentiation, and increase blood calcium levels in rats more effectively than 1,25(OH)2D3. In this study, to further investigate the usefulness of 2α-substituted vitamin D derivatives, we examined the effects of O2C3, O1C3, and their derivatives on VDR activity in cells and mouse tissues and on osteoblast differentiation of dedifferentiated fat (DFAT) cells, a cell type with potential therapeutic application in regenerative medicine. In cell culture experiments using kidney-derived HEK293 cells, intestinal mucosa-derived CaCO2 cells, and osteoblast-derived MG63 cells, and in mouse experiments, O2C2, O2C3, O1C3, and O1C4 had a weaker effect than or equivalent effect to 1,25(OH)2D3 in VDR transactivation and induction of the VDR target gene CYP24A1, but they enhanced osteoblast differentiation in DFAT cells equally to or more effectively than 1,25(OH)2D3. In long-term treatment with the compound without the medium change (7 days), the derivatives enhanced osteoblast differentiation more effectively than 1,25(OH)2D3. O2C3 and O1C3 were more stable than 1,25(OH)2D3 in DFAT cell culture. These results indicate that 2α-substituted vitamin D derivatives, such as inactivation-resistant O2C3 and O1C3, are more effective than 1,25(OH)2D3 in osteoblast differentiation of DFAT cells, suggesting potential roles in regenerative medicine with DFAT cells and other multipotent cells.

The Role of Intestinal Cytochrome P450s in Vitamin D Metabolism.

Vitamin D hydroxylation in the liver/kidney results in conversion to its physiologically active form of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. 1,25(OH)2D3 controls gene expression through the nuclear vitamin D receptor (VDR) mainly expressed in intestinal epithelial cells. Cytochrome P450 (CYP) 24A1 is a catabolic enzyme expressed in the kidneys. Interestingly, a recently identified mutation in another CYP enzyme, CYP3A4 (gain-of-function), caused type III vitamin D-dependent rickets. CYP3A are also expressed in the intestine, but their hydroxylation activities towards vitamin D substrates are unknown. We evaluated CYP3A or CYP24A1 activities on vitamin D action in cultured cells. In addition, we examined the expression level and regulation of CYP enzymes in intestines from mice. The expression of CYP3A or CYP24A1 significantly reduced 1,25(OH)2D3-VDRE activity. Moreover, in mice, Cyp24a1 mRNA was significantly induced by 1,25(OH)2D3 in the intestine, but a mature form (approximately 55 kDa protein) was also expressed in mitochondria and induced by 1,25(OH)2D3, and this mitochondrial enzyme appears to hydroxylate 25OHD3 to 24,25(OH)2D3. Thus, CYP3A or CYP24A1 could locally attenuate 25OHD3 or 1,25(OH)2D3 action, and we suggest the small intestine is both a vitamin D target tissue, as well as a newly recognized vitamin D-metabolizing tissue.

Vitamin D-metabolizing enzyme CYP24A1 affects oncogenic behaviors of oral squamous cell carcinoma and its prognostic implication.

Vitamin D is an essential molecule for cellular homeostasis, playing a critical role in cell fate decisions including cell proliferation, differentiation, and viability. Accumulating evidence has revealed that expression of the vitamin D-metabolizing enzyme CYP24A1 is dysregulated in different types of human malignancy. CYP24A1 has been shown to be involved in the oncogenic property of a variety of carcinoma cells. However, the pathological relevance of CYP24A1 expression level in human oral malignancy remains to be clarified. In the present study, suppression of CYP24A1 expression in oral squamous cell carcinoma (OSCC) cells increased cell proliferation, invasive activity, colony formation efficacy, and tumor growth in vivo. In addition, knockout of CYP24A1 expression inhibited cell death induced by two different types of anticancer drugs, i.e., fluorouracil and cisplatin. Gene clustering by RNA-sequence analysis revealed that several signaling molecules associated with MYC are involved in CYP24A1-mediated oncogenic behaviors. Furthermore, decreased expression level of CYP24A1 was observed in 124/204 cases (61%) of OSCC and was shown to be associated with short relapse-free and overall survival periods. The results showed that a low expression level of CYP24A1 promotes the oncogenic activity of OSCC and is significantly associated with poor prognosis in patients with this malignancy.

Electro-scalp acupuncture regulates the expression of CYP27a1/b1, CYP24a and related inflammatory cytokines in ischemic cortex of rats with middle cerebral artery occlusion. / ç”µå¤´é’ˆè°ƒæŽ§å¤§è„‘ä¸­åŠ¨è„‰æ “å¡žå¤§é¼ ç¼ºè¡€å¤§è„‘çš®å±‚åŒºCYP27a1/b1、CYP24aåŠç›¸å ³ç‚Žæ€§ç»†èƒžå› å­è¡¨è¾¾çš„ç ”ç©¶.

OBJECTIVES: To observe the effect of electro-scalp acupuncture (ESA) on the expression of cytochrome P450a1/b1 (CYP27a1/b1), cytochrome P45024a (CYP24a), signal transducer and activator of transcription (STAT)4, STAT6, tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-4 in ischemic cerebral cortex of rats with acute ischemic stroke, so as to explore its mechanism in alleviating inflammatory reaction of ischemic stroke. METHODS: Sixty SD rats were randomly divided into sham-operation, model, vitamin D3 and ESA groups, with 15 rats in each group. The middle cerebral artery occlusion rat model was established with thread ligation according to Zea-Longa's method. Rats in the vitamin D3 group were given 1, 25-VitD3 solution (3 ng·100 g-1·d-1) by gavage, once daily for 7 days. Rats in the ESA group were treated at bilateral anterior parietotemporal slash (MS6) with ESA (2 Hz/100 Hz, 1 mA), 30 min a day for 7 days. Before and after interventions, the neurological deficit score and neurobehavioral score were evaluated. TTC staining was used to detect the volume of cerebral infarction in rats. The positive expressions of CYP24a, CYP27a1 and CYP27b1 in the cerebral cortex of ischemic area were detected by immunofluorescence. The mRNA expressions of STAT4 and STAT6 in the cerebral cortex of ischemic area were detected by quantitative real-time PCR. The protein expression levels of TNF-α, IL-1ß and IL-4 in the cerebral cortex of ischemic area were detected by Western blot. RESULTS: Compared with the sham-operation group, the neurological deficit score, neurobehavioral score, the percentage of cerebral infarction volume, the positive expression level of CYP24a and mRNA expression level of STAT4, protein expression levels of TNF-α and IL-1ß in cerebral cortex were increased (P<0.01), while the positive expression levels of CYP27a1/b1 and STAT6 mRNA, protein expression level of IL-4 were decreased (P<0.01) in the model group. After the treatment and compared with the model group, the neurological deficit score, neurobehavioral score, the percentage of cerebral infarction volume, the positive expression level of CYP24a and mRNA expression level of STAT4, protein expression levels of TNF-α and IL-1ß in cerebral cortex were decreased (P<0.01), while the positive expression levels of CYP27a1/b1 and STAT6 mRNA expression level, protein expression level of IL-4 were increased (P<0.01) in the ESA and vitamin D3 groups. CONCLUSIONS: ESA can alleviate the inflammatory response in ischemic stroke, which maybe related to its function in regulating the balance between CYP27a1/b1 and CYP24a, converting vitamin D into active vitamin D3, inhibiting vitamin D3 degradation, and regulating Th1/Th2 balance.

Accumulated LPS induced by colitis altered the activities of vitamin D-metabolizing hydroxylases and decreased the generation of 25-hydroxyvitamin D.

It is generally accepted that low vitamin D (VD) levels are associated with a high prevalence factor for Inflammatory bowel disease (IBD). IBD patients have observed higher levels of lipopolysaccharide (LPS), ALT, and AST than healthy people. Gut-derived LPS causes inflammatory injury in the liver and kidney. The VD-metabolizing mechanism is involved in the liver and kidney, which means IBD might impact VD metabolism. However, whether IBD affects VD metabolism has not been studied. In vitro LPS resulted in decreased CYP2R1 in liver cells as well as decreased CYP27B1 and increased CYP24A1 in kidney cells, revealing that LPS changed the activities of several hydroxylases. Mice with acute colitis had an increased LPS in serum and liver with mild hepatic injuries, while mice with chronic colitis had a significant elevation of LPS in serum, liver, and kidney with hepatorenal injuries. Thus, the liver hydroxylase for VD metabolism would be the first to be affected in IBD. Consequently, serum 25-hydroxyvitamin D declined dramatically with a significant elevation of 24,25-dihydroxyvitamin D and 1,24,25-trihydroxyvitamin D. Unchanged serum levels of 1,25-dihydroxyvitamin D might be the result of other factors in vivo. In acute colitis, a small dosage (4 IU/day) of cholecalciferol could protect the colon, decrease the serum level of LPS, and finally increase serum 25-hydroxyvitamin D. However, this improvement of cholecalciferol was fading in chronic colitis. These results suggested that VD supplementations for preventing and curing IBD in the clinic should consider hepatorenal hydroxylases and be employed as soon as possible for a better outcome.

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.

Cellular responses to silencing of PDIA3 (protein disulphide-isomerase A3): Effects on proliferation, migration, and genes in control of active vitamin D.

The active form of vitamin D, 1,25-dihydroxyvitamin D3, is known to act via VDR (vitamin D receptor), affecting several physiological processes. In addition, PDIA3 (protein disulphide-isomerase A3) has been associated with some of the functions of 1,25-dihydroxyvitamin D3. In the present study we used siRNA-mediated silencing of PDIA3 in osteosarcoma and prostate carcinoma cell lines to examine the role(s) of PDIA3 for 1,25-dihydroxyvitamin D3-dependent responses. PDIA3 silencing affected VDR target genes and significantly altered the 1,25-dihydroxyvitamin D3-dependent induction of CYP24A1, essential for elimination of excess 1,25-dihydroxyvitamin D3. Also, PDIA3 silencing significantly altered migration and proliferation in prostate PC3 cells, independently of 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 increased thermostability of PDIA3 in cellular thermal shift assay, supporting functional interaction between PDIA3 and 1,25-dihydroxyvitamin D3-dependent pathways. In summary, our data link PDIA3 to 1,25-dihydroxyvitamin D3-mediated signalling, underline and extend its role in proliferation and reveal a novel function in maintenance of 1,25-dihydroxyvitamin D3 levels.

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.

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.

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.