C-25 hydroxylation of 1alpha,24(R)-dihydroxyvitamin D3 is catalyzed by 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1): metabolism studies with human keratinocytes and rat recombinant CYP24A1

Recently, 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) has been shown to catalyze not only hydroxylation at C-24 but also hydroxylations at C-23 and C-26 of the secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). It remains to be determined whether CYP24A1 has the ability to hydroxylate vitamin D3 compounds at C-25. 1alpha,24(R)-dihydroxyvitamin D3 (1alpha,24(R)(OH)2D3) is a non-25-hydroxylated synthetic vitamin D3 analog that is presently being used as an antipsoriatic drug. In the present study, we investigated the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes in order to examine the ability of CYP24A1 to hydroxylate 1alpha,24(R)(OH)2D3 at C-25. The results indicated that keratinocytes metabolize 1alpha,24(R)(OH)2D3 into several previously known both 25-hydroxylated and non-25-hydroxylated metabolites along with two new metabolites, namely 1alpha,23,24(OH)3D3 and 1alpha,24(OH)2-23-oxo-D3. Production of the metabolites including the 25-hydroxylated ones was detectable only when CYP24A1 activity was induced in keratinocytes 1alpha,25(OH)2D3. This finding provided indirect evidence to indicate that CYP24A1 catalyzes C-25 hydroxylation of 1alpha,24(R)(OH)2D3. The final proof for this finding was obtained through our metabolism studies using highly purified recombinant rat CYP24A1 in a reconstituted system. Incubation of this system with 1alpha,24(R)(OH)2D3 resulted in the production of both 25-hydroxylated and non-25-hydroxylated metabolites. Thus, in our present study, we identified CYP24A1 as the main enzyme responsible for the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes, and provided unequivocal evidence to indicate that the multicatalytic enzyme CYP24A1 has the ability to hydroxylate 1alpha,24(R)(OH)2D3 at C-25.     

25(OH)D3 and 1,25(OH)2D3 serum concentration and breast tissue expression of 1alpha-hydroxylase, 24-hydroxylase and Vitamin D receptor in women with and without breast cancer

1,25(OH)2D3 is an antiproliferative agent that may inhibit proliferation of breast cancer (BC) cells in vitro and BC development in animals. Epidemiological studies have shown a high incidence of BC in people less exposed to solar rays. To unravel the role of Vitamin D3 in BC patients, we have investigated serum levels of 25(OH)D3 and its active form 1,25(OH)2D3 as well as tissue expression of 1alpha-hydroxylase, 24-hydroxylase, and Vitamin D-receptor (VDR), determined by semiquantitative RT-PCR, in 88 Brazilian BC patients and 35 women without cancer (submitted to mammoplasties or resection of benign lesions). Median age of women with and without cancer was 51 and 46 years, respectively, and the majority of BC patients were classified as clinical stage II (67%). Although no differences in 25(OH)D3 serum concentration were found, 1,25(OH)2D3 (40+/-21 pg/ml) levels in BC patients were lower than in women without cancer (53+/-23). Our results indicate that 24-hydroxylase, VDR and 1alpha-hydroxylase mRNA tissue expression is similar in both groups and no correlation between 24-hydroxylase, 1alpha-hydroxylase, and VDR expression in breast tumors was found. A low 1,25(OH)2D3 serum concentration seems to be associated to breast cancer, however, the mechanism involved in this regulation is still unclear.     

Clonal differences in expression of 25-hydroxyvitamin D(3)-1alpha-hydroxylase,of 25-hydroxyvitamin D(3)-24-hydroxylase,and of the vitamin D receptor in human colon carcinoma cells: effects of epidermal growth factor and 1alpha,25-dihydroxyvitamin D(3)

Human colon carcinoma cells express 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP27B1) and thus produce the vitamin D receptor (VDR) ligand 1alpha,25-dihydroxyvitamin D(3) (1,25-D3), which can be metabolized by 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24). Expression of VDR, CYP27B1, and CYP24 determines the efficacy of the antimitotic action of 1,25-D3 and is distinctly related to the degree of differentiation of cancerous lesions. In the present study we addressed the question of whether the effects of epidermal growth factor (EGF) and of 1,25-D3 on VDR, CYP27B1, and CYP24 gene expression in human colon carcinoma cell lines also depend on the degree of cellular differentiation. We were able to show that slowly dividing, highly differentiated Caco-2/15 cells responded in a dose-dependent manner to both EGF and 1,25-D3 by up-regulation of VDR and CYP27B1 expression, whereas in highly proliferative, less differentiated cell lines, such as Caco-2/AQ and COGA-1A and -1E, negative regulation was observed. CYP24 mRNA was inducible in all clones by 1,25-D3 but not by EGF. From the observed clonal differences in the regulatory effects of EGF and 1,25-D3 on VDR and CYP27B1 gene expression we suggest that VDR-mediated growth inhibition by 1,25-D3 would be efficient only in highly differentiated carcinomas even when under mitogenic stimulation by EGF.     

Association of 25-Hydroxyvitamin D status and genetic variation in the vitamin D metabolic pathway with FEV1 in the Framingham Heart Study

Background

Vitamin D is associated with lung function in cross-sectional studies, and vitamin D inadequacy is hypothesized to play a role in the pathogenesis of chronic obstructive pulmonary disease. Further data are needed to clarify the relation between vitamin D status, genetic variation in vitamin D metabolic genes, and cross-sectional and longitudinal changes in lung function in healthy adults.

Methods

We estimated the association between serum 25-hydroxyvitamin D [25(OH)D] and cross-sectional forced expiratory volume in the first second (FEV₁) in Framingham Heart Study (FHS) Offspring and Third Generation participants and the association between serum 25(OH)D and longitudinal change in FEV₁ in Third Generation participants using linear mixed-effects models. Using a gene-based approach, we investigated the association between 241 SNPs in 6 select vitamin D metabolic genes in relation to longitudinal change in FEV₁ in Offspring participants and pursued replication of these findings in a meta-analyzed set of 4 independent cohorts.

Results

We found a positive cross-sectional association between 25(OH)D and FEV₁ in FHS Offspring and Third Generation participants (P = 0.004). There was little or no association between 25(OH)D and longitudinal change in FEV₁ in Third Generation participants (P = 0.97). In Offspring participants, the CYP2R1 gene, hypothesized to influence usual serum 25(OH)D status, was associated with longitudinal change in FEV₁ (gene-based P < 0.05). The most significantly associated SNP from CYP2R1 had a consistent direction of association with FEV₁ in the meta-analyzed set of replication cohorts, but the association did not reach statistical significance thresholds (P = 0.09).

Conclusions

Serum 25(OH)D status was associated with cross-sectional FEV₁, but not longitudinal change in FEV₁. The inconsistent associations may be driven by differences in the groups studied. CYP2R1 demonstrated a gene-based association with longitudinal change in FEV₁ and is a promising candidate gene for further studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0238-y) contains supplementary material, which is available to authorized users.     

Autoradiographic studies with 3H 1,25 (OH)2 vitamin D3 and 3H 25 (OH) vitamin D3 in rat parathyroid glands

Summary After injection of radiolabeled 1,25 (OH)₂ vitamin D₃, nuclear concentration of radioactivity is observed in parenchymal cells of the parathyroid gland in pregnant, adult male, and 10-day male neonatal rats. In competition studies with unlabeled 1,25 (OH)₂ vitamin D₃, but not with 25 (OH) vitamin D₃, nuclear uptake is prevented. Experiments with ³H 25 (OH) vitamin D₃, in contrast to ³H 1,25 (OH)₂ vitamin D₃, do not show nuclear concentration in cells of the parathyroid. The results of the autoradiographic studies suggest the presence of receptors for a direct effect of 1,25 (OH)₂ vitamin D₃ on the parathyroid gland for modulation of parathyroid hormone secretion.     

Generation of a homology model for the human cytochrome P450, CYP24A1, and the testing of putative substrate binding residues by site-directed mutagenesis and enzyme activity studies

A systematic analysis of conserved H-bonding patterns and tertiary structural motifs from 13 crystal structures was used to create a homology model for the human multicatalytic cytochrome P450, CYP24A1, involved in catabolism of 1alpha,25-dihydroxyvitamin D3. The substrate was docked in the active site and used to identify potential substrate contact residues in the B' helix, B'/C loop, F-helix and the beta-5 hairpin. Seven CYP24A1 mutants were created and studied by mammalian cell transfection and CYP24A1 activity assay. Mutants showed reduced metabolic rates and altered metabolite patterns compared to wild-type. We conclude that: Ile-131 positions substrate via A-ring and cis-triene contacts; Trp-134 and Gly-499 are determinants of substrate access; Leu-148 contacts the substrate side-chain; Met-246 is important in mediating regioselectivity. Our findings validate the new model of CYP24A1, which can now be used to predict structural modifications for rational vitamin D drug design.     

Hybrid homology modeling and mutational analysis of cytochrome P450C24A1 (CYP24A1) of the Vitamin D pathway: insights into substrate specificity and membrane bound structure-function

Cytochrome P450C24A1 (CYP24A1), a peripheral inner mitochondrial membrane hemoprotein and candidate oncogene, regulates the side-chain metabolism and biological function of vitamin D and many of its related analog drugs. Rational mutational analysis of rat CYP24A1 based on hybrid (2C5/BM-3) homology modeling and affinity labeling studies clarified the role of key domains (N-terminus, A', A, and F-helices, beta3a strand, and beta5 hairpin) in substrate binding and catalysis. The scope of our study was limited by an inability to purify stable mutant enzyme targeting soluble domains (B', G, and I-helices) and suggested greater conformational flexibility among CYP24A1's membrane-associated domains. The most notable mutants developed by modeling were V391T and I500A, which displayed defective-binding function and profound metabolic defects for 25-hydroxylated vitamin D3 substrates similar to a non-functional F-helix mutant (F249T) that we previously reported. Val-391 (beta3a strand) and Ile-500 (beta5 hairpin) are modeled to interact with Phe-249 (F-helix) in a hydrophobic cluster that directs substrate-binding events through interactions with the vitamin D cis-triene moiety. Prior affinity labeling studies identified an amino-terminal residue (Ser-57) as a putative active-site residue that interacts with the 3beta-OH group of the vitamin D A-ring. Studies with 3-epi and 3-deoxy-1,25(OH)2D3 analogs confirmed interactions between the 3beta-OH group and Ser-57 effect substrate recognition and trafficking while establishing that the trans conformation of A-ring hydroxyl groups (1alpha and 3beta) is obligate for high-affinity binding to rat CYP24A1. Our work suggests that CYP24A1's amphipathic nature allows for monotopic membrane insertion, whereby a pw2d-like substrate access channel is formed to shuttle secosteroid substrate from the membrane to the active-site. We hypothesize that CYP24A1 has evolved a unique amino-terminal membrane-binding motif that contributes to substrate specificity and docking through coordinated interactions with the vitamin D A-ring.     

Removal of C-ring from the CD-ring skeleton of 1alpha,25-dihydroxyvitamin D3 does not alter its target tissue metabolism significantly

It is now well established that 1alpha,25(OH)2D3 is metabolized in its target tissues through the modifications of both side chain and A-ring. The C-24 oxidation pathway is the side chain modification pathway through which 1alpha,25(OH)2D3 is metabolized into calcitroic acid. The C-3 epimerization pathway is the A-ring modification pathway through which 1alpha,25(OH)2D3 is metabolized into 1alpha,25(OH)2-3-epi-D3. During the past two decades, a great number of vitamin D analogs were synthesized by altering the structure of both side chain and A-ring of 1alpha,25(OH)2D3 with the aim to generate novel vitamin D compounds that inhibit proliferation and induce differentiation of various types of normal and cancer cells without causing significant hypercalcemia. Previously, we used some of these analogs as molecular probes to examine how changes in 1alpha,25(OH)2D3 structure would affect its target tissue metabolism. Recently, several nonsteroidal analogs of 1alpha,25(OH)2D3 with unique biological activity profiles were synthesized. Two of the analogs, SL 117 and WU 515 lack the C-ring of the CD-ring skeleton of 1alpha,25(OH)2D3. SL 117 contains the same side chain as that of 1alpha,25(OH)2D3, while WU 515 contains an altered side chain with a 23-yne modification combined with hexafluorination at C-26 and C-27. Presently, it is unknown how the removal of C-ring from the CD-ring skeleton of 1alpha,25(OH)2D3 would affect its target tissue metabolism. In the present study, we compared the metabolic fate of SL 117 and WU 515 with that of 1alpha,25(OH)2D3 in both the isolated perfused rat kidney, which expresses only the C-24 oxidation pathway and rat osteosarcoma cells (UMR 106), which express both the C-24 oxidation and C-3 epimerization pathways. The results of our present study indicate that SL 117 is metabolized like 1alpha,25(OH)2D3, into polar metabolites via the C-24 oxidation pathway in both rat kidney and UMR 106 cells. As expected, WU 515 with altered side chain structure is not metabolized via the C-24 oxidation pathway. Unlike in rat kidney, both SL 117 and WU 515 are also metabolized into less polar metabolites in UMR 106 cells. These metabolites displayed GC and MS characteristics consistent with A-ring epimerization and were putatively assigned as C-3 epimers of SL 117 and WU 515. In summary, we report that removal of the C-ring from the CD-ring skeleton of 1alpha,25(OH)2D3 does not alter its target tissue metabolism significantly.     

Toxicity and antineoplastic effect of (24R)-1,24-dihydroxyvitamin D3 (PRI-2191)

Many efforts have been made to obtain active and less toxic Vitamin D analogs for new clinical applications. The results of previous studies demonstrated the efficacy and safety of topical treatment of psoriasis with one of these analogs, 1,24-dihydroxyvitamin D(3), tacalcitol (1,24-(OH)(2)D(3)). In the present study, we evaluated the toxicity and antitumor effect of this analog. Lethal toxicity of 1,24-(OH)(2)D(3) after s.c. injection was significantly lower than that of calcitriol. No significant differences were observed in the toxicity of the analogs when administered p.o. Calcium levels in the serum of mice treated with calcitriol were significantly higher (111%) than those in mice treated with 1,24-(OH)(2)D(3) (89%) at 5 day after the first s.c. (10 microg/kg/day) administration in comparison to the control (healthy, untreated animals). Oral administration increased the calcium level by 78% for calcitriol and only to 47% over the control for 1,24-(OH)(2)D(3). Parallel administration of clodronate prevented the calcitriol- and 1,24-(OH)(2)D(3)-induced lethal toxicity and also prevented increase in calcium levels. Single therapy with calcitriol did not affect tumor growth in the 16/C mouse mammary cancer model. In contrary, 1,24-(OH)(2)D(3) alone reduced tumor volume to 41% of control. Cisplatin alone did not affect growth of 16/C tumor in these conditions. The growth of tumors in the presence of cisplatin was inhibited by 1,24-(OH)(2)D(3) but not by calcitriol. Interestingly, the inhibition of tumor growth in cisplatin-treated mice by 1,24-(OH)(2)D(3) was greater, than that observed in mice treated with this analog alone. In conclusion, 1,24-(OH)(2)D(3) revealed higher antitumor and lower calcemic activity and toxicity than calcitriol. Application of biphosphonates along with Vitamin D analogs is sufficient to overcome the calcemic and toxic side effects of the proposed treatment.     

1alpha,25(OH)2 vitamin D3 actions on ion channels in osteoblasts

Membrane-initiated cellular responses to steroids include modulation of ion channel activities via signal transduction pathways. However, the molecular mechanisms involved in nongenomic actions remain only partially understood. Our research has focused on the rapid effects of 1alpha,25(OH)(2) Vitamin D(3) [1,25D] on L-type Ca(2+) [L-Ca] and DIDS-sensitive Cl(-) channels in osteoblasts. Physiological nanomolar concentrations of hormonally active 1,25D promote rapid (1-5 min) potentiation of outward Cl(-) currents in osteosarcoma ROS 17/2.8 cells and mouse primary osteoblasts. In addition, 1,25D increases inward barium currents through L-Ca channels at low depolarizing potentials within seconds in a fashion similar to the 1,4-dihydropyridine [DHP] agonist Bay K8644. We found that second messenger cAMP is involved in 1,25D potentiation of Cl(-) and Ca(2+) channels. Nongenomic 1,25D effects on ion channel activities in osteoblasts appear to involve different mechanisms that include a possible direct interaction with the L-Ca channel molecule, on one hand, and signaling through the cAMP pathway, on the other. Rapid 1,25D actions on Cl(-) and Ca(2+) currents seem to couple to secretory activities in osteoblasts, thus contributing to bone mass formation.     

Phosphate uptake in chick kidney cells: effects of 1,25(OH)2D3 and 24,25(OH)2D3

Phosphate homeostasis is controlled in part by absorption from the intestine, and reabsorption in the kidney. While the effect of Vitamin D metabolites on enterocytes is well documented, in the current study we assess selected responses in primary cultures of kidney cells. Time course studies revealed a rapid stimulation of phosphate uptake in cells treated with 1,25(OH)(2)D(3), relative to controls. Dose-response studies indicated a biphasic curve with optimal stimulation at 300 pM 1,25(OH)(2)D(3) and inhibition at 600 pM seco-steroid. Antibody 099--against the 1,25D(3)-MARRS receptor - abolished stimulation by the steroid hormone. Moreover, phosphate uptake was mediated by the protein kinase C pathway. The metabolite 24,25(OH)(2)D(3), which was found to inhibit the rapid stimulation of phosphate uptake in intestinal cells, had a parallel effect in cultured kidney cells. Finally, the 24,25(OH)(2)D(3) binding protein, catalase, was assessed for longer term down regulation. In both intestinal epithelial cells and kidney cells incubated with 24,25(OH)(2)D(3) for 5-24h, both the specific activity of the enzyme and protein levels were decreased relative to controls, while 1,25(OH)(2)D(3) increased both parameters over the same time periods. We conclude that the Vitamin D metabolites have similar effects in both kidney and intestine, and that 24,25(OH)(2)D(3) may have effects at the level of gene expression.     

Characterization of transgenic rats constitutively expressing vitamin D-24-hydroxylase gene

Vitamin D-24-hydroxylase (CYP24) is one of the enzymes responsible for vitamin D metabolism. CYP24 catalyzes the conversion of 25-hydroxyvitamin D(3) [25(OH)D(3)] to 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)] in the kidney. CYP24 is also involved in the breakdown of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], the active form of vitamin D(3). In this study, we generated transgenic (Tg) rats constitutively expressing CYP24 gene to investigate the biological role of CYP24 in vivo. Surprisingly, the Tg rats showed a significantly low level of plasma 24,25(OH)(2)D(3). Furthermore, the Tg rats developed albuminuria and hyperlipidemia shortly after weaning. The plasma lipid profile revealed that all lipoprotein fractions were elevated in the Tg rats. Also, the Tg rats showed atherosclerotic lesions in the aorta, which greatly progressed with high-fat and high-cholesterol feeding. These unexpected results suggest that CYP24 is involved in functions other than the regulation of vitamin D metabolism.     

Chemical synthesis of 20S-hydroxyvitamin D3, which shows antiproliferative activity

20S-hydroxyvitamin D3 (20S-(OH)D3), an in vitro product of vitamin D3 metabolism by the cytochrome P450scc, was recently isolated, identified and shown to possess antiproliferative activity without inducing hypercalcemia. The enzymatic production of 20S-(OH)D3 is tedious, expensive, and cannot meet the requirements for extensive chemical and biological studies. Here we report for the first time the chemical synthesis of 20S-(OH)D3 which exhibited biological properties characteristic of the P450scc-generated compound. Specifically, it was hydroxylated to 20,23-dihydroxyvitamin D3 and 17,20-dihydroxyvitamin D3 by P450scc and was converted to 1α,20-dihydroxyvitamin D3 by CYP27B1. It inhibited proliferation of human epidermal keratinocytes with lower potency than 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) in normal epidermal human keratinocytes, but with equal potency in immortalized HaCaT keratinocytes. It also stimulated VDR gene expression with similar potency to 1,25(OH)2D3, and stimulated involucrin (a marker of differentiation) and CYP24 gene expression, showing a lower potency for the latter gene than 1,25(OH)2D3. Testing performed with hamster melanoma cells demonstrated a dose-dependent inhibition of cell proliferation and colony forming capabilities similar or more pronounced than those of 1,25(OH)2D3. Thus, we have developed a chemical method for the synthesis of 20S-(OH)D3, which will allow the preparation of a series of 20S-(OH)D3 analogs to study structure-activity relationships to further optimize this class of compound for therapeutic use.     

Isolation and identification of 2alpha,25-dihydroxyvitamin D3, a new metabolite from Pseudonocardia autotrophica 100U-19 cells incubated with Vitamin D3

Pseudonocardia autotrophica converted Vitamin D(3) to 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3). The hydroxylation of Vitamin D(3) with P. autotrophica was enhanced by the addition of cyclodextrin. In this microbial hydroxylation, a new Vitamin D(3) metabolite was observed in the reaction mixture of P. autotrophica and Vitamin D(3), and was isolated in a pure form by several steps of chromatography. The structure of the new metabolite was determined to be 2alpha,25-dihydroxyvitamin D(3) by UV, NMR and mass spectroscopic analyses. Biological evaluation of the new metabolite was conducted by means of several experiments.     

Real-life use of vitamin D3-fortified bread and milk during a winter season: the effects of CYP2R1 and GC genes on 25-hydroxyvitamin D concentrations in Danish families,the VitmaD study

Common genetic variants rs10741657 and rs10766197 in CYP2R1 and rs4588 and rs842999 in GC and a combined genetic risk score (GRS) of these four variants influence late summer 25-hydroxyvitamin D (25(OH)D) concentrations. The objectives were to identify those who are most at risk of developing low vitamin D status during winter and to assess whether vitamin D₃-fortified bread and milk will increase 25(OH)D concentrations in those with genetically determined low 25(OH)D concentrations at late summer. We used data from the VitmaD study. Participants were allocated to either vitamin D₃-fortified bread and milk or non-fortified bread and milk during winter. In the fortification group, CYP2R1 (rs10741657) and GC (rs4588 and rs842999) were statistically significantly associated with winter 25(OH)D concentrations and CYP2R1 (rs10766197) was borderline significant. There was a negative linear trend between 25(OH)D concentrations and carriage of 0–8 risk alleles (p < 0.0001). No association was found for the control group (p = 0.1428). There was a significant positive linear relationship between different quintiles of total vitamin D intake and the increase in 25(OH)D concentrations among carriers of 0–2 (p = 0.0012), 3 (p = 0.0001), 4 (p = 0.0118) or 5 (p = 0.0029) risk alleles, but not among carriers of 6–8 risk alleles (p = 0.1051). Carriers of a high GRS were more prone to be vitamin D deficient compared to carriers of a low GRS. Furthermore, rs4588-AA carriers have a low but very stable 25(OH)D concentration, and interestingly, also low PTH level.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0413-7) contains supplementary material, which is available to authorized users.