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Altered Pharmacokinetics of 1,25-Dihydroxyvitamin D₃ and 25-Hydroxyvitamin D₃ in the Blood and Tissues of the 25-Hydroxyvitamin D-24-Hydroxylase (Cyp24a1) Null Mouse

Department of Biochemistry, Queen’s University (S.M., V.B., G.J.), Kingston, Ontario, Canada K7L 3N6; Genetics Unit, Shriners Hospital for Children (A.A., R.S.-A.), Montréal, Québec, Canada H3G 1A6; Departments of Medicine, Surgery, and Human Genetics, McGill University (R.S.-A.), Montréal, Québec, Canada H3A 2T5; and Endocrine Unit, Massachusetts General Hospital, Harvard Medical School (Y.S., M.B.D.), Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Dr. Glenville Jones, Department of Biochemistry, Queen’s University, Kingston Ontario, Canada K7L 3N6. E-mail: gj1{at}post.queensu.ca.

	Abstract

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The 25-hydroxyvitamin D-24-hydroxylase (CYP24A1) plays an important role in regulating concentrations of both the precursor 25-hydroxyvitamin D₃ [25(OH)D₃] and the hormone 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. Previous studies suggest that Cyp24a1-null mice cannot clear exogenous 1,25(OH)₂D₃ efficiently. Here, we examined the metabolic clearance in Cyp24a1-null mice in vivo and in vitro using a physiological dose of [1ß-³H]1,25(OH)₂D₃ or [26,27-methyl-³H]25(OH)D₃. Cyp24a1-null mice showed difficulty in eliminating [1ß-³H]1,25(OH)₂D₃ from the bloodstream and tissues over a 96-h time course, whereas heterozygotic mice eliminated the hormone within 6–12 h, although there was clearance of labeled hormone into water-soluble products involving liver in both genotypes. RT-PCR showed that Cyp24a1-null mice have decreased expression of 25-hydroxyvitamin D-1-hydroxylase that must play a role in their survival. After the administration of [26,27-methyl-³H]25(OH)D₃, Cyp24a1-null mice showed higher [26,27-methyl-³H]25(OH)D₃ levels and no [26,27-methyl-³H]24,25(OH)₂D₃ formation, whereas heterozygotic mice showed significant [26,27-methyl-³H]24,25(OH)₂D₃ production. Based upon in vitro experiments, keratinocytes from Cyp24a1-null mice fail to synthesize [1ß-³H]calcitroic acid from [1ß-³H]1,25(OH)₂D₃ or [26,27-methyl-³H]24,25(OH)₂D₃ from [26,27-methyl-³H]25(OH)D₃ as do control mice, confirming the target cell catabolic role of CYP24A1 in these processes. Finally, the role of vitamin D receptor (VDR) in the vitamin D catabolic cascade was examined using VDR-null mice. Keratinocytes from VDR-null mice failed to metabolize [1ß-³H]1,25(OH)₂D₃ confirming the importance of vitamin D-inducible, VDR-mediated, C24 oxidation pathway in target cells. These results suggest that the absence of CYP24A1 or VDR retards catabolism of 1,25(OH)₂D₃ and 25(OH)D₃, reinforcing the physiological importance of CYP24A1 in vitamin D homeostasis.

	Introduction

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THE SEQUENCING OF the human genome has led to the realization that among a pool of 60 total cytochrome P450s (CYPs),¹ there are three known and possibly other uncharacterized vitamin D-related CYPs dedicated to the metabolism of vitamin D. The key enzymes in vitamin D metabolism are the hepatic vitamin D-25-hydroxylase [CYP27A1 and possibly CYP2R1 (1)], renal 25-hydroxyvitamin D-1-hydroxylase (CYP27B1), and 25-hydroxyvitamin D-24-hydroxylase (CYP24A1). Biological activation of vitamin D occurs through the sequential hydroxylation catalyzed by CYP27A1 or CYP2R1 and CYP27B1, resulting in the formation of the active form of vitamin D₃, 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] (2, 3, 4). CYP24A1 is the enzyme acting on 1,25(OH)₂D₃ to initiate degradation of this potent metabolite and is tightly regulated. This enzyme is distributed in classical vitamin D target tissues, including kidney, intestine, bone, and skin. In target cells, 1,25(OH)₂D₃ has the ability to induce its own degradation by up-regulating CYP24A1 expression to attenuate 1,25(OH)₂D₃ action or prevent vitamin D toxicity (i.e. hypercalcemia) (5, 6, 7). CYP24A1 was discovered as the mitochondrial enzyme responsible for the hydroxylation at C-24 in the metabolism of 1,25(OH)₂D₃ and 25-hydroxyvitamin D₃ [25(OH)D₃] (8, 9, 10). C-24 oxidation represents the predominant catabolic pathway for 1,25(OH)₂D₃ in which sequential C-24 hydroxylation, C-24 ketonization, and C-23 hydroxylation, followed by oxidative cleavage to form tetranor-1,23(OH)₂D₃ and calcitroic acid, liberate an inactive, water-soluble product that is excreted in bile (5, 11). The secondary pathway involves C-26 hydroxylation, C-23 hydroxylation, and lactonization to form (23S,25R)-1,25(OH)₂D₃-26,23-lactone (12, 13). Both pathways have also been shown to participate in the metabolism of 25(OH)D₃ (13). Studies of the recombinant CYP24A1 protein produced in Escherichia coli (14, 15), insect (16), and mammalian (17) cells have revealed that CYP24A1 exerts multicatalytic activity, facilitating sequential oxidation of C-23, C-24, and C-26 hydroxylation and side-chain cleavage.

The role of CYP24A1 has been established to be inactivation of both 1,25(OH)₂D₃ and 25(OH)D₃ (5, 6, 13). Most evidence from kinetic analysis supports the concept that 1,25(OH)₂D₃, rather than 25(OH)D₃, is the preferred substrate for CYP24A1 (18) and that this enzyme plays an important role in regulating the intracellular concentration as well as the half-life of 1,25(OH)₂D₃ in the body. Recently, this was also confirmed by the generation of a Cyp24a1 knockout mouse model (19). Initial reports suggest that Cyp24a1-null mice show prolonged elevations in plasma 1,25(OH)₂D₃ levels after exogenous 1,25(OH)₂D₃ administration. These findings are consistent with the poor viability of the Cyp24a1-null mice and its tendency to hypercalcemia and nephrocalcinosis during the neonatal period. Interestingly, Cyp24a1 mutant animals that survived past weaning showed lower baseline levels of circulating 1,25(OH)₂D₃ than wild-type controls. This fact raises questions about the physiological role of CYP24A1 in the vitamin D metabolism. It has been postulated that these animals may use an alternative pathway of 1,25(OH)₂D₃ catabolism to regulate circulating levels of the hormone.

The action of 1,25(OH)₂D₃ in target cells is mediated by the vitamin D receptor (VDR), which binds to vitamin D response elements in the promoter of genes. Two vitamin D response elements in the CYP24A1 promoter are responsible for the VDR-mediated up-regulation of CYP24A1 gene transcription (20, 21). In mice lacking the VDR gene, highly elevated plasma levels of 1,25(OH)₂D₃ were measured due to the impaired ligand-dependent activation of Cyp24a1 gene transcription and inadequate catabolic degradation of 1,25(OH)₂D₃ (22, 23). Hypocalcemia and a loss of VDR-mediated gene expression caused Cyp24a1 expression to be low in such animals. Thus, because of the inducible nature of vitamin D-target cell CYP24A1, the VDR-null mouse represents the equivalent of a partial Cyp24a1 knockout mouse model. Accordingly, a comparison of CYP24A1 enzyme activity in VDR-null mice and Cyp24a1-null mice would allow us to understand the roles of both VDR and CYP24A1 in vitamin D signaling and catabolic cascade.

The development of novel gene expression systems for CYPs has allowed examination of some long-standing questions in vitamin D metabolism. In the present study we focused on 1) elucidation of the overall importance of CYP24A1 in 1,25(OH)₂D₃ and 25(OH)D₃ clearance, 2) direct comparison of the metabolism of 1,25(OH)₂D₃ and 25(OH)D₃ in the Cyp24a1-null mouse in vivo and in vitro, and 3) elucidation of the role of VDR in 1,25(OH)₂D₃ metabolism using keratinocytes from the VDR-null mouse in vitro. The results confirm that ablation of either the Cyp24a1 or VDR gene profoundly retards metabolism of 1,25(OH)₂D₃ and 25(OH)D₃ and that the surviving Cyp24a1-null mice owe their survival in part to down-regulation of Cyp27b1 expression.

	Materials and Methods

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Materials
1,25(OH)₂D₃, 25(OH)D₃, 1,24,25(OH)₃D₃, tetranor-1,23(OH)₂D₃, 24,25(OH)₂D₃, and 1(OH)D₃ were gifts from Drs. M. Calverley and A. M. Kissmeyer (Leo Pharmaceutical Products, Ballerup, Denmark). (23S,25R)-1,25(OH)₂D₃-26,23-lactone was a gift from Dr. S. Ishizuka (Teijin, Japan). [26,27-Methyl-³H]25(OH)D₃ (30 Ci/mmol) was purchased from Amersham Biosciences (Little Chalfont, UK). [1ß-³H]1,25(OH)₂D₃ (50 Ci/mmol) and calcitroic acid were synthesized by methods described previously (5). MEM, trypsin, and antibiotic/antimyotic were purchased from Invitrogen Life Technologies (Burlington, Canada). Fetal calf serum (FCS) was purchased from ICN (Costa Mesa, CA), and BSA was obtained from Roche (Laval, Canada). Chelex 100 was purchased from Bio-Rad Laboratories (Hercules, CA). Collagen (Vitrogen 100) was purchased from Cohesion Technologies, Inc. (Palo Alto, CA). Epidermal growth factor (EGF) and N'N-diphenyl-p-phenylenediamine (DPPD) were supplied by Sigma-Aldrich Corp. (St. Louis, MO). Liquid scintillation cocktail (Ready Flow III) for on-line radioactivity detection was purchased from Beckman Coulter, Inc. (Fullerton, CA). All organic solvents were of HPLC grade and were purchased from Caledon Laboratories Ltd. (Georgetown, Canada).

Animals
All studies performed were reviewed and approved by the institutional animal care and use committees. Animals were maintained in a virus- and parasite-free barrier facility and exposed to a 12-h light, 12-h dark cycle. Mice lacking Cyp24a1 were obtained as previously described (19). Genotyping for the Cyp24a1 mutation was performed by Southern blotting of BamHI digests using an EcoRI-PstI genomic probe downstream of the region of homology. The 12-kb wild-type allele and the 8.5-kb targeted allele were identified. Mice lacking VDR were obtained as previously described (22). VDR-null mice were genotyped by PCR analysis of tail DNA using primers specific for the neomycin resistance gene and the second zinc finger of the VDR.

In vivo metabolism study in Cyp24a1-null mice
For the 1,25(OH)₂D₃ metabolism study, 6-month-old Cyp24a^–/– homozygotes and their Cyp24a^+/– littermates received a single iv injection of [1ß-³H]1,25(OH)₂D₃ (1 µCi; 8 ng) in 100 µl of a vehicle containing ethanol/mouse serum (1:9). At 3, 6, 24, 48, and 96 h postinjection, blood was taken by cardiac puncture, and liver and kidneys were collected. For the 25(OH)D₃ metabolism study, 6-month-old Cyp24a1^–/– homozygotes and their Cyp24a^+/– littermates received a single iv injection of [26,27-methyl-³H]25(OH)D₃ (5 µCi; 67 ng) in 100 µl of a vehicle containing ethanol/mouse serum (1:9). At 6, 24, and 48 h postinjection, blood was taken by cardiac puncture and liver, kidneys and intestine were collected. Plasma was obtained by centrifugation at 3000 rpm for 15 min. Liver, kidneys, and intestine were frozen in liquid N₂ and stored at –20 C.

Isolation and preparation of primary keratinocytes
Primary keratinocytes were isolated from 2- to 3-d-old Cyp24a1-null mice and control littermates by a trypsin floating procedure as described previously (24). Briefly, the skin was dissected from the trunk of mice and floated on 0.25% trypsin for 16–20 h at 4 C. The epidermis was then peeled off the dermis, minced with scissors, and stirred in MEM with 4% (vol/vol) Chelex-treated FCS, EGF (10 ng/ml), 1% (vol/vol) antibiotic/antimycotic, and 0.05 mM CaCl₂ (low calcium medium) for 1 h at 4 C. The resultant cell suspension was then filtered through a 70-µm pore size filter (Falcon, BD Biosciences, Franklin Lakes, NJ) to remove clumps of cells and debris. The keratinocytes were pelleted by centrifugation for 5 min at room temperature and plated in low calcium medium in collagen-coated, 60-mm dishes. Cells were maintained at 37 C in a humidified atmosphere of 5% CO₂ in air. At 80–90% confluence, cells were reseeded at 1.8 x 10⁵ cells/well in six-well plates in low calcium medium and grown to 80% confluence before addition of substrate.

In vitro metabolism study of 1,25(OH)₂D₃ and 25(OH)D₃ in Cyp24a1-null mouse keratinocytes
Primary keratinocytes obtained from each mouse were treated with 10 nM 1,25(OH)₂D₃ (in ethanol) to induce the transcription of Cyp24a1. After 18 h, the cells were rinsed twice with PBS and incubated with 0.25 µCi [1ß-³H]1,25(OH)₂D₃ (2.1 ng; 2.5 nM) in 2 ml low calcium MEM supplemented with 1% (wt/vol) BSA, 10 ng/ml EGF, 1% (vol/vol) antibiotic/antimycotic, and 100 µM DPPD for 24 h at 37 C. For the time-course study of 25(OH)D₃, mouse primary keratinocytes that were not previously induced with 10 nM 1,25(OH)₂D₃ were incubated with 0.5 µCi [26,27-methyl-³H]25(OH)D₃ (6.6 ng; 16.6 nM) in 2 ml low calcium MEM supplemented with 1% (wt/vol) BSA, 10 ng/ml EGF, 1% (vol/vol) antibiotic/antimycotic, and 100 µM DPPD for 0, 1, 3, 6, and 12 h at 37 C.

In vitro metabolism study in VDR-null mice keratinocytes
Mouse primary keratinocytes from VDR-null and wild-type mice were plated at 2 x 10⁶ cells/plate in P-100 plates and grown to 80% confluence in low calcium MEM with 4% (vol/vol) Chelex-treated FCS, 10 ng/ml EGF, and 1% (vol/vol) antibiotic/antimycotic. Cells were incubated with 0.5 µCi [1ß-³H]1,25(OH)₂D₃ (4.16 ng; 2.5 nM) including 1 µM 1,25(OH)₂D₃ or 10 µM 1,25(OH)₂D₃ in 4 ml low calcium MEM supplemented with 1% (wt/vol) BSA, 10 ng/ml EGF, 1% (vol/vol) antibiotic/antimycotic, and 100 µM DPPD for 24 h at 37 C.

RT-PCR analysis of CYP27B1 transcripts
The RT-PCR protocol used total RNA prepared with the TRIzol reagent (Invitrogen Life Technologies, Inc., Gaithersburg, MD) from the kidneys of 4-month-old Cyp24a1^+/– and Cyp24a1^–/– animals (25). The RT reaction contained oligo(deoxythymidine) and SuperScript II reverse transcriptase (Invitrogen Life Technologies, Inc.). PCR amplification used the following primers for Cyp27b1: 5'-CTGCGAGGAGGGGTAAGGTGTT-3' and 5'-GGAAACGGGGGAGGGGA-3'. Amplification was for 28 cycles. Control ß-actin amplification reactions used the primer pair 5'-GCTGCGTGTGGCCCCTAGG-3' and 5'-CAAGAAGGAAGGCTGGAAAAGAG-3'. Amplimers were detected by ethidium bromide staining of agarose electrophoresis gels.

Lipid extraction
Before the extraction, plasma was adjusted up to a volume of 1 ml with ice-cold buffered 0.9% saline, and tissues were homogenated in ice-cold buffered 0.9% saline (4 ml/g tissue). An internal standard of 2 µg 1(OH)D₃ was added to blood and tissue homogenate samples to assess recovery during the extraction process. For the cell culture, incubations were terminated at designated time points with methanol. All samples were extracted using a modification of the method of Bligh and Dyer (26), in which chloroform was replaced by methylene chloride. The aqueous layer was reextracted with methylene chloride and 0.1% glacial acetic acid to isolate the water-soluble metabolites. After the extraction, aliquots of organic and aqueous fractions were subjected to liquid scintillation counter (LC1800, Beckman Coulter, Inc., Fullerton, CA). All samples were dried down under a stream of prepurified N₂ at 37 C, resuspended, and stored in ethanol.

HPLC
Metabolites from in vivo or in vitro study were analyzed by either straight phase (organic-soluble metabolites) or reverse phase (water-soluble metabolites) HPLC using an Alliance 2695 Separations Module and a model 996 photodiode array detector (Waters Associates, Milford, MA). Radiolabeled organic- or water-soluble metabolites were detected using a RadioFlow Detector (LB 509, EG&G Berthold, Bad Wildbad, Germany). The organic phase extracts were dried down, redissolved, and separated on a Zorbax SIL column (3 µm; 62 x 80 mm; Agilent Technologies, Palo Alto, CA) with a hexane/isopropanol/methanol (HIM; 91%:7%:2%, vol/vol/vol) isocratic system at a flow rate of 1.0 ml/min. The water-soluble metabolites were separated on a Zorbax SB-C₁₈ column (5 µm; 4.6 x 150 mm; Agilent Technologies) with a linear gradient solvent system, which started at 69%:30%:1% (vol/vol/vol) water/acetonitrile/10% acetic acid and was brought to 0%/99%/1% (vol/vol/vol) water/acetonitrile/10% acetic acid by 30 min at a flow rate of 1.0 ml/min. Metabolites were identified based on the characteristic vitamin D cis-triene chromophore (_max = 265 nm; _min = 228 nm; _max/min = 1.75). Data analysis was performed with the aid of Millennium³² chromatographic software (Waters Associates, Milford, MA).

Statistical analysis
The significance of the difference between the means of the two groups was analyzed by t test, and differences were taken as statistically significant at P < 0.001 (***), P < 0.01 (**), or P < 0.05 (*).

	Results

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Metabolism of 1,25(OH)₂D₃ in Cyp24a1-null mice in vivo
Initial studies focused on the metabolic clearance and blood and tissue levels of [1ß-³H]1,25(OH)₂D₃ and its circulating metabolites over a 96-h period in Cyp24a1^–/– homozygotic mice and their Cyp24a1^+/– littermates after a single iv injection of 1 µCi [1ß-³H]1,25(OH)₂D₃. The results shown in Fig. 1 indicate that the marked differences in lipid-soluble radioactivity between Cyp24a1^+/– control and Cyp24a1^–/– mice were evident by as early as 6 h. Even at 6 h, organic fractions from blood, liver, and kidney tissues from Cyp24a1^–/– mice showed approximately 2-fold higher [1ß-³H]1,25(OH)₂D₃ levels than Cyp24a1^+/– controls. By 96 h this difference became 20-fold for blood, 7-fold for liver, and 50-fold for kidney. [1ß-³H]1,25(OH)₂D₃ was still identifiable in blood, liver, and kidney of Cyp24a1^–/– mice at 96 h. The half-life (t_1/2) of [1ß-³H]1,25(OH)₂D₃ in Cyp24a1^–/– mice was significantly longer than that in Cyp24a1^+/– controls in blood (t_1/2, 39 h vs. 4 h), liver (t_1/2, 16 h vs. 5 h), and kidney (t_1/2, 12 h vs. 5 h). The lipid-soluble ³H radioactivity remained mainly in the form of 1,25(OH)₂D₃, except in blood and liver of Cyp24a1^+/– mice at early time points, where [1ß-³H]1,25(OH)₂D₃-26,23-lactone was also detectable. [1ß-³H]1,25(OH)₂D₃-26,23-lactone was completely absent from the blood of Cyp24a1^–/– mice despite the fact that a measurable peak of [1ß-³H]1,25(OH)₂D₃ was always greater in chromatograms of Cyp24a1^–/– mice than in Cyp24a1^+/– mice. As a result of these initial studies, subsequent experiments performed by our group emphasized the need to explore the water-soluble metabolites in more detail.

View larger version (19K): [in this window] [in a new window]   FIG. 1. Levels of [1ß-3H]1,25(OH)2D3 in Cyp24a1–/–and Cyp24a1+/– mice after a single bolus dose. [1ß-3H]1,25(OH)2D3 levels in blood, liver, and kidney in Cyp24a1-null mice were compared with heterozygotic littermates at 6, 24, 48, and 96 h after a single iv administration of 1 µCi [1ß-3H]1,25(OH)2D3. Lipids from the extraction were separated on Zorbax SIL (3 µm; 62 x 80 mm) column using the solvent HIM (91:7:2) at a flow rate of 1.0 ml/min. Radioactivity measured using radioisotope detector and identified as [1ß-3H]1,25(OH)2D3 from its retention time is plotted. Each point represents the mean ± SE of three animals. Differences were statistically significant as indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared with Cyp24a1+/– mice by t test).

View this table: [in this window] [in a new window]   TABLE 1. Time course of clearance of 1,25(OH)D2D3 in Cyp24a1–/– and Cyp24a1+/– mice after the administration of 1 µCi [1ß-3H]1,25(OH)2D3

View larger version (25K): [in this window] [in a new window]   FIG. 2. Cyp27b1 [25-hydroxyvitamin D-1-hydroxylase (1-OHase)] expression in the kidney from Cyp24a1–/– and Cyp24a1+/– mice. The expression of Cyp27b1 was analyzed by RT-PCR using total RNA prepared from kidney tissue of 4-month-old Cyp24a1–/– and Cyp24a1+/– mice. The conditions for the RT-PCR were described in Materials and Methods. The 451-bp Cyp27b1 amplimer and the 522-bp ß-actin amplimer are shown. Three animals were probed for each genotype; genotypes are identified below the figure. Note the dramatic reduction in Cyp27b1 expression in Cyp24a1–/– mice.

View larger version (17K): [in this window] [in a new window]   FIG. 3. HPLC profile of metabolites of [1ß-3H]1,25(OH)2D3 generated by incubation of primary keratinocyte from Cyp24a1–/– and Cyp24a1+/– mice with 0.25 µCi [1ß-3H] 1,25(OH)2D3 for 24 h after induction with 10 nM 1,25(OH)2D3. Lipid-soluble metabolites (A) were separated on a Zorbax SIL (3 µm; 62 x 80 mm) column using the solvent HIM (91:7:2) using a flow rate of 1.0 ml/min. Water-soluble metabolites (B) were separated on a Zorbax SCB18 (5 µm; 4.6 x 150 mm) column using a linear water-acetonitrile gradient system from 30–100% acetonitrile with 0.1% acetic acid over 30 min at a flow rate of 1.0 ml/min. Metabolites of [1ß-3H]1,25(OH)2D3 were identified by flow radioactivity detection using synthetic standards. The experiment was performed using five to six animals from each group, and a representative chromatogram is shown.

View larger version (25K): [in this window] [in a new window]   FIG. 4. Levels of [26,27-methyl-3H]25(OH)D3 in Cyp24a1–/– and Cyp241a+/– mice after a single bolus dose. [26,27-Methyl-3H]25(OH)D3 levels in blood, liver, kidney, and intestine from Cyp24a1-null mice were compared with their heterozygotic littermates at 6, 24, and 48 h after a single iv administration with 5 µCi [26,27-methyl-3H]25(OH)D3. Lipids from the extraction were separated on a Zorbax SIL (3 µm; 62 x 80 mm) column using the solvent HIM (91:7:2) at a flow rate of 1.0 ml/min. Radioactivity measured using flow radioisotope detector and identified as [26,27-methyl-3H]25(OH)D3 from its retention time is plotted. Each data point is the mean ± SE of two animals. Differences were statistically significant as indicated: *, P < 0.05; **, P < 0.005 (compared with Cyp24a1+/– mice by the t test).

View larger version (19K): [in this window] [in a new window]   FIG. 5. HPLC profiles of blood and tissue samples extracted from Cyp24a1–/– and Cyp24a1+/– mice at 48 h after iv administration of [26,27-methyl-3H]25(OH)D3. Lipid extracts from blood, liver, kidney, and intestine (4.5 x 105 dpm of each) were chromatographed on a Zorbax SIL (3 µm; 62 x 80 mm) column using the solvent HIM (91:7:2) at a flow rate of 1.0 ml/min. Metabolites of [26,27-methyl-3H]25(OH)D3 were identified by retention time with synthetic standards after detection using a flow radioisotope detector. The experiment was performed using two animals, and a typical chromatogram from each group is depicted.

View larger version (19K): [in this window] [in a new window]   FIG. 6. HPLC profiles of metabolites of [26,27-methyl-3H]25(OH)D3 generated in primary keratinocyte culture of Cyp24a1–/– and Cyp24a1+/– mice. Keratinocytes were incubated with 0.5 µCi [26,27-methyl-3H]25(OH)D3 for 0, 1, 3, 6, and 12 h. Lipid-soluble metabolites were separated on a Zorbax SIL (3 µm; 62 x 80 mm) column using the solvent HIM (91:7:2) at a flow rate of 1.0 ml/min. Metabolites of [26,27-methyl-3H]25(OH)D3 were identified by flow radioactivity detection using synthetic standards. The experiment was performed using three to seven animals in each group, and representative chromatograms are shown for 0, 6, and 12 h.

Metabolism of 1,25(OH)₂D₃ in VDR-null mice keratinocytes in vitro

View larger version (24K): [in this window] [in a new window]   FIG. 7. HPLC profile of metabolites of [1ß-3H]1,25(OH)2D3 generated in primary keratinocyte culture of VDR-null and wild-type mice. Keratinocytes were incubated with 0.5 µCi [1ß-3H] 1,25(OH)2D3 (specific activity, 125 mCi/mmol) for 24 h. Lipid-soluble metabolites were separated on a Zorbax SIL (3 µm; 62 x 80 mm) column using the solvent HIM (91:7:2) at a flow rate of 1.0 ml/min. Metabolites of [1ß-3H]1,25(OH)2D3 were identified by flow radioactivity detection using synthetic standards. The radioactivity recovered in the aqueous fraction, measured by liquid scintillation counter, is shown in the inset.

	Discussion

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Not only do Cyp24a1-null mice lack 24-hydroxylated metabolites, but they also appear to lack 1,25(OH)₂D₃-26,23-lactone despite the fact that the concentration of the [1ß-³H]1,25(OH)₂D₃ hormone was always higher in Cyp24a1^–/– mice than in Cyp24a1^+/– mice, and thus substrate was presumably not rate-limiting. This result reinforces the view that CYP24A1 is also responsible for hydroxylation of 1,25(OH)₂D₃ at C-23 and C-26. In contrast, the fact that half of the homologous mutant animals survived into adulthood despite the inactivation of the Cyp24a1 in mice that caused lethality in their littermates suggests the existence of an alternative pathway of inactivation of 1,25(OH)₂D₃ or some other form of compensation, such as altered rate of production of 1,25(OH)₂D₃. No detectable [1ß-³H]3-epi-1,25(OH)₂D₃ (27) was found in mice in vivo or in keratinocytes in vitro even though HPLC conditions would have resolved it from the natural hormone. This suggests that 3-epimerization of 1,25(OH)₂D₃ is not the alternative pathway of inactivation responsible for eventual clearance of [1ß-³H]1,25(OH)₂D₃ in Cyp24a1^–/– mice. In contrast, a fraction of the radioactivity partitions into the aqueous layer during excretion at all time points, especially in the liver in both genotypes, and although the exact amount is difficult to quantify because the aqueous fraction always contains some unchanged [1ß-³H]1,25(OH)₂D₃, this aqueous phase radioactivity most likely represents some other uncharacterized, water-soluble product. This is suggestive of an alternative undefined route of degradation, possibly involving glucuronidation or another route of conjugation (28, 29, 30). Indeed, because the relative contribution of the water-soluble fraction increases with time in the Cyp24a1^+/– animals but not in the Cyp24a1^–/– animals, the results support the hypothesis that a good portion of the clearance is CYP24A1 dependent.

However, one alternative hypothesis is that surviving animals down-regulate the synthesis of 1,25(OH)₂D₃. Indeed, we have previously reported that Cyp24a1^–/– mice that survive past weaning (i.e. 50% of the mutant population) show baseline circulating levels of 1,25(OH)₂D₃ that are 2.8-fold lower than those in age-matched heterozygous littermates (19). This obviously allows these Cyp24a1^–/– mutant mice to survive without significant impairment of mineral ion homeostasis, at least in the absence of a challenge to normocalcemia (such as pregnancy, for example) (19). It is noteworthy that Cyp24a1 mutant mice showed significantly increased expression levels of vitamin D-dependent genes involves in VDR in the kidney at the basal point compared with heterozygote controls, and the expression levels in these mice did not respond to 1,25(OH)₂D₃ administration (19). Iida et al. (31) suggested that regulation of VDR expression would allow reciprocal control of CYP27B1 and CYP24A1 activity in renal proximal tubules. We postulated that it might be possible that up-regulated VDR in the kidney of Cyp24a1^–/– mice acts on Cyp27b1 expression to suppress the production of 1,25(OH)₂D₃, resulting in lower circulating levels of 1,25(OH)₂D₃. The RT-PCR data presented here suggest that surviving Cyp24a1^+/– animals show reduced expression of Cyp27b1 mRNA compared with their Cyp24a1^+/– littermates at 4 months of age. Accordingly, these results confirm that pathways of 1,25(OH)₂D₃ elimination are through the inducible expression of CYP24A1 in vivo and possibly an as yet to be defined basal clearance pathway, but that Cyp24a1^+/– animals also adapt to vitamin D intoxication by down-regulation of 1,25(OH)₂D₃ production.

After the administration of [26,27-methyl-³H]25(OH)D₃, higher residual [26,27-methyl-³H]25(OH)D₃ levels and no [26,27-methyl-³H]24,25(OH)₂D₃ formation were observed in blood and tissues (liver, kidney, and intestine) of Cyp24a1^–/– mice over 48 h. In contrast to our observations with [1ß-³H]1,25(OH)₂D₃, metabolic clearance of [26,27-methyl-³H]25(OH)D₃ was slower in both Cyp24a1^–/– and Cyp24a1^+/– mice. Administered [26,27-methyl-³H]25(OH)D₃ stayed mostly in the blood over the full 48-h time course, with limited uptake by tissues in either genotype. These data are consistent with the longer half-life reported for 25(OH)D₃ and reflect the fact that the clearance of 25(OH)D₃ is greatly affected by its sequestration by the plasma carrier protein, DBP, that is synthesized in the liver. Although there was a slightly higher level of tissue total radioactivity in the tissues of Cyp24a1^–/– mice compared with Cyp24a1^+/– mice, there was a marked increase in residual [26,27-methyl-³H]25(OH)D₃ levels in the blood and tissues (liver, kidney, and intestine) of Cyp24a1^–/– mice compared with Cyp24a1^+/– littermates, reflecting the reduced rate of metabolism. Indeed, chromatography showed the complete absence of 24-hydroxylated metabolites in the blood and tissues of Cyp24a1^–/– mice. These results indicate the critical importance of CYP24A1 in the catabolism of 25(OH)D₃.

Another way to investigate the actions of CYP24A1 in the natural setting using Cyp24a1^–/– mice is to perform in vitro studies to avoid the complexity of the in vivo setting. Keratinocytes were chosen as target cells to examine the physiological role of CYP24A1 in the catabolic cascade of vitamin D in vitro. When primary neonatal keratinocytes were prepared from Cyp24a1^–/– and Cyp24a1^+/– mice, the findings were essentially the same as those of the in vivo study, demonstrating in particular the lack of calcitroic acid production and 24,25(OH)₂D₃ formation in keratinocytes from Cyp24a1^–/– mice. Previous studies demonstrated that skin is capable of activating vitamin D₃ and 25(OH)D₃ through sequential hydroxylations, including 1-hydroxylation, and the resulting 1,25(OH)₂D₃ plays a role in epidermal homeostasis in normal and diseased skin (32, 33, 34). Interestingly, in our hands these Cyp24a1^–/– keratinocytes failed to produce detectable amounts of 1,25(OH)₂D₃ production even though they were cultured under low calcium conditions, and the masking action of CYP24A1 was absent. The lack of 1,25(OH)₂D₃ production was possibly due to the presence of FCS and its associated vitamin D metabolites in the culture medium. The fact that none of the metabolites, including C-23/C-26 oxidation products, was detected from the incubation with keratinocytes from Cyp24a1^–/– mice leads to the conclusion that CYP24A1 plays multiple roles in 1,25(OH)₂D₃ and 25(OH)D₃ catabolism. To date, our results suggest that CYP24A1 is the main enzyme required for the catabolism of 1,25(OH)₂D₃ and 25(OH)D₃, although it might be prudent to not completely exclude alternative pathways until we have fully defined the nature of water-soluble biliary metabolites in the Cyp24a1^–/– mice.

The results from the keratinocyte study of 1,25(OH)₂D₃ in VDR^–/– mice indicate that Cyp24a1 expression is mediated through VDR. When primary keratinocytes were incubated with a physiological concentration of 1,25(OH)₂D₃, VDR^+/+ mice demonstrated a series of metabolites of 1,25(OH)₂D₃ through the C-24/C-23 oxidation pathway. However, keratinocytes from VDR^–/– mice showed a complete block of target cell metabolism of 1,25(OH)₂D₃ (as shown in Fig. 7). The difference illustrates the highly inducible nature of target cell Cyp24a1. Even when substrate was increased to pharmacological concentrations, little metabolism was observed in cells from VDR^–/– mice compared with VDR^+/+ mice (data not shown). One published in vivo study using VDR-null mice (35) demonstrated that the degradation of 1,25(OH)₂D₃ in VDR-null mice was substantially reduced compared with that in wild-type control mice. Interestingly, unlike the in vitro study, 1,25,26(OH)₃D₃ was found in plasma, kidney, and liver of VDR-null mice (35). This suggests that an enzyme that catalyzes the 26-hydroxylation of 1,25(OH)₂D₃, that would be independent of VDR stimulation, may exist in vivo_. Recently, several liver cytochrome P450 isoforms, including CYP2R1, CYP2J3, CYP3A4, as well as CYP27A1, have been shown to possess vitamin D side-chain hydroxylation activity, and one wonders if this activity can be directed at the C26 position as well as the C25 position, as is currently claimed (1, 36, 37).

In conclusion, the Cyp24a1-null mouse is a valuable model, with potential to establish the pharmacological as well as the physiological role of CYP24A1. The availability of the Cyp24a1-null mouse also opens up new approaches to determine the degree of involvement of CYP24A1 in vitamin D analog metabolism.

	Footnotes

 
This work was supported by Operating and Equipment Grants MT-9475 and MMA-38116 from the Canadian Institutes of Health Research (to G.J.), a research grant from the Shriners of North America (to R.S.-A.), and National Institutes of Health Grant R01-DK-46974 (to M.B.D.). Aspects of this work were presented to the 22nd Annual Meeting of the American Society for Bone and Mineral Research in Toronto (38 39 ).

First Published Online October 21, 2004

¹ Cytochrome P450 nomenclature was followed by the Cytochrome P450 Homepage at http://drnelson.utmem.edu/humP450.aln.html.

Abbreviations: CYP24A1, 25-Hydroxyvitamin D-24-hydroxylase; CYP27A1, vitamin D-25-hydroxylase; CYP27B1, 25-hydroxyvitamin D-1-hydroxylase; DBP, vitamin D-binding protein; DPPD, N',N-diphenyl-p-phenylenediamine; EGF, epidermal growth factor; FCS, fetal calf serum; HIM, hexane/isopropanol/methanol; 1,25(OH)₂D₃, 1,25-dihydroxyvitamin D₃; 25(OH)D₃, 25-hydroxyvitamin D₃; 24,25(OH)₂D₃, 24(R),25-dihydroxyvitamin D₃; 1,24,25(OH)₃D₃, 1,24(R),25-trihydroxyvitamin D₃; 1,25(OH)₂D₃-26,23-lactone, (23S,25R)-1,25-dihydroxyvitamin D₃-26,23-lactone; 1(OH)D₃, 1-hydroxyvitamin D₃; VDR, vitamin D receptor.

Received August 23, 2004.

Accepted for publication October 14, 2004.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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