1alpha ,25-Dihydroxyvitamin D3-24-Hydroxylase (CYP24) Hydroxylates the Carbon at the End of the Side Chain (C-26) of the C-24-fluorinated Analog of 1alpha ,25-Dihydroxyvitamin D3*

(Received for publication, October 22, 1996, and in revised form, March 17, 1997)

Yoichi Miyamoto Dagger §, Toshimasa Shinki Dagger , Keiko Yamamoto §, Yoshihiko Ohyama par , Hiroshi Iwasaki , Ryuzo Hosotani , Toshio Kasama , Hiroaki Takayama **, Sachiko Yamada § and Tatsuo Suda Dagger Dagger Dagger

From the Dagger  Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, the § Division of Molecular Biology, Institute for Medical and Dental Engineering, Tokyo Medical and Dental University, 2-3-10 Surugadai, Kanda, Chiyoda-ku, Tokyo 101, the  Tsukuba Research Laboratory, NOF Corporation, 5-10 Tokodai, Tsukuba-shi, Ibaraki 300-21, the par  Graduate Department of Gene Science, Faculty of Science, Hiroshima University, 1-3-1 Kagamiya, Higashi-Hiroshima, Hiroshima 724, and the ** Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The sequential oxidation and cleavage of the side chain of 1alpha ,25-dihydroxyvitamin D3 (1alpha ,25(OH)2D3) initiated by the hydroxylation at C-24 is considered to be the major pathway of this hormone in the target cell metabolism. In this study, we examined renal metabolism of a synthetic analog of 1alpha ,25(OH)2D3, 24,24-difluoro-1alpha ,25-dihydroxyvitamin D3 (F2-1alpha ,25(OH)2D3), C-24 of which was designed to resist metabolic hydroxylation. When kidney homogenates prepared from 1alpha ,25(OH)2D3-supplemented rats were incubated with F2-1alpha ,25(OH)2D3, it was mainly converted to a more polar metabolite. We isolated and unequivocally identified the metabolite as 24,24-difluoro-1alpha ,25,26-trihydroxyvitamin D3 (F2-1alpha ,25,26(OH)3D3) by ultraviolet absorption spectrometry, frit-fast atom bombardment liquid chromatography/mass spectroscopy analysis, and direct comparison with chemically synthesized F2-1alpha ,25,26(OH)3D3. Metabolism of F2-1alpha ,25(OH)2D3 into F2-1alpha ,25,26(OH)3D3 by kidney homogenates was induced by the prior administration of 1alpha ,25(OH)2D3 into rats. The C-24 oxidation of 1alpha ,25(OH)2D3 in renal homogenates was inhibited by F2-1alpha ,25(OH)2D3 in a concentration-dependent manner. Moreover, F2-1alpha ,25,26(OH)3D3 was formed in ROS17/2.8 cells transfected with a plasmid expressing 1alpha ,25(OH)2D3-24-hydroxylase (CYP24) but not in the cells transfected with that expressing vitamin D3-25-hydroxylase (CYP27) or containing inverted CYP27 cDNA. These results show that CYP24 catalyzes not only hydroxylation at C-24 and C-23 of 1alpha ,25(OH)2D3 but also at C-26 of F2-1alpha ,25(OH)2D3, indicating that this enzyme has a broader substrate specificity of the hydroxylation sites than previously considered.


INTRODUCTION

Metabolic inactivation of 1alpha ,25-dihydroxyvitamin D3 (1alpha ,25(OH)2D3),1 the biologically active metabolite of vitamin D3, in its target cells is initiated by side chain hydroxylation at C-23, C-24, and C-26 (1-4). Of these hydroxylation sites, it is now accepted that the sequential oxidation and cleavage of the side chain initiated by the hydroxylation at C-24 catalyzed by mitochondrial 1alpha ,25(OH)2D3-24-hydroxylase (CYP24) is the major pathway by which the hormone is inactivated (5). Because transcription of the CYP24 gene is highly up-regulated by 1alpha ,25(OH)2D3 in its target cells (6-8), CYP24 is regarded as the key enzyme for the breakdown of the hormone (9). Metabolism of 1alpha ,25(OH)2D3 initiated by C-23 hydroxylation is induced by 1alpha ,25(OH)2D3 itself (10), and recombinant human CYP24 also catalyzes C-23 hydroxylation of 25-hydroxyvitamin D3 (25(OH)D3) to yield 23S,25-dihydroxyvitamin D3 (23S,25(OH)2D3) (11). Therefore, it is likely that CYP24 initiates both C-24 and C-23 hydroxylation pathways of 1alpha ,25(OH)2D3. In contrast, mitochondrial vitamin D-25-hydroxylase (CYP27) catalyzes the hydroxylation at C-25 and C-26 of vitamin D3 and 1alpha -hydroxyvitamin D3 (1alpha (OH)D3) (12), but it is not clear whether or not this enzyme hydroxylates C-26 of 1alpha ,25(OH)2D3. Recently, a model for the mechanism of the hydroxylation site selection by CYP24 and CYP27 was proposed. This model postulates that CYP24 directs its hydroxylation site(s) by the distance of C-24 and C-23 from the vitamin D ring structure and that CYP27 does so by the distance between the hydroxylation sites and the end of the side chain (13).

Of numerous synthetic analogs of 1alpha ,25(OH)2D3, 24,24-difluoro-1alpha ,25-dihydroxyvitamin D3 (F2-1alpha ,25(OH)2D3) (14) was the first that had a higher biological activity than the parental 1alpha ,25(OH)2D3 (15-17). Although the biological activity of F2-1alpha ,25(OH)2D3 was higher, the binding affinity of this analog to VDR was almost identical to that of 1alpha ,25(OH)2D3 (16, 17). It is accepted that the resistance of the C-F bond at C-24 of this analog to metabolic inactivation contributes to its higher biological activity. The metabolic fate of F2-1alpha ,25(OH)2D3, however, has not yet been clarified. We recently reported that F2-1alpha ,25(OH)2D3 is metabolized into a more polar compound(s) in rat osteoblastic ROB-C26 cells (17). The metabolism was initiated after transcription of the CYP24 gene, which was induced by the substrate, F2-1alpha ,25(OH)2D3 itself (17).

In this study, we examined whether CYP24 can metabolize vitamin D analogs, the C-24 of which is resistant to hydroxylation. We identified 24,24-difluoro-1alpha ,25,26-trihydroxyvitamin D3 (F2-1alpha ,25,26(OH)3D3) as a major metabolite of F2-1alpha ,25(OH)2D3. Moreover, the enzyme catalyzing the conversion of F2-1alpha ,25(OH)2D3 into F2-1alpha ,25,26(OH)3D3 was CYP24.


EXPERIMENTAL PROCEDURES

Materials

All chemicals were of highly purified analytical grade. F2-1alpha ,25(OH)2D3 (18), (25R)- and (25S)-F2-1alpha ,25,26(OH)3D32 were synthesized in our laboratory. The 1alpha ,25-dihydroxy[1beta -3H]vitamin D3 (1alpha ,25(OH)2[1beta -3H]D3) (specific activity, 202 GBq/mmol) and 24,24-difluoro-1alpha ,25-dihydroxy[1beta -3H]vitamin D3 (F2-1alpha ,25(OH)2[1beta -3H]D3) (specific activity, 396 GBq/mmol) were prepared by Daiichi Pure Chemicals, Co., Ltd. (Tokyo, Japan) as described (19). 1alpha ,25(OH)2D3 was purchased from Wako Pure Chemicals (Osaka, Japan). 1alpha ,24R,25-trihydroxyvitamin D3 (1alpha ,24R,25(OH)3D3) was a gift from Kureha Chemical Industries (Tokyo, Japan). [alpha -32P]dCTP (specific activity, 111 TBq/mmol) was from Amersham International plc (Buckinghamshire, UK). Guanidine isothiocyanate was from Fluka Biochemika (Buchs, Switzerland). A random primer labeling kit was obtained from Takara Biochemicals (Kyoto, Japan). The expression plasmid for CYP24 (pSVL-CYP24) (20) was prepared in our laboratory. The cDNA for CYP27 (21), the expression plasmid for CYP27 (pSVL-CYP27) (21), and the plasmid harboring inverted CYP27 cDNA (pSVL-CYP27(-)) were gifts from Dr. E. Usui (Hiroshima University, Hiroshima, Japan). The cDNA for mouse beta -tubulin was a gift of Dr. N. J. Cowan (New York University, New York, NY). A rat osteosarcoma cell line, ROS17/2.8, was donated by Dr. M. Noda (Tokyo Medical and Dental University, Tokyo, Japan).

Metabolism of F2-1alpha ,25(OH)2D3 by Rat Renal Homogenates

1 µg of 1alpha ,25(OH)2D3 or vehicle (ethanol) was injected intravenously to 6-week-old male Sprague Dawley rats. 8 h later, the rats were killed by decapitation, and then the kidneys were quickly removed, rinsed, and homogenized in 9 volumes of ice-cold 15 mM Tris acetate buffer (pH 7.4) containing 0.19 M sucrose, 2 mM magnesium acetate, and 5 mM sodium succinate (22). Homogenates (3 ml) were incubated with 0.25 nM F2-1alpha ,25(OH)2[1beta -3H]D3 (specific activity, 3.5 GBq/mmol) for 15 min at 37 °C under oxygen with constant shaking (22), and then total lipids were extracted by the method of Bligh and Dyer (23). The chloroform phase was analyzed by a straight phase HPLC under the following conditions: column, Finepak SIL 4.6 × 250 mm (Jasco, Tokyo, Japan); mobile phase, n-hexane/chloroform/methanol, 10:2.5:1.5; flow rate, 1 ml/min, or by reverse phase HPLC under the following conditions: column, J' sphere ODS-AM 4.6 × 150 mm (YMC Co. Ltd., Kyoto, Japan); mobile phase, a linear concentration gradient of acetonitrile (40 to 100% in 30 min); flow rate, 1 ml/min. The eluate was collected in 30- or 18-s fractions. The radioactivity in each fraction was measured in a scintillation fluid (OPTI-FLUOR-O, Packard Instrument Company, Meriden, CT or ACS II, Amersham International plc).

Metabolism of 1alpha ,25(OH)2D3 by Rat Renal Homogenates

The renal homogenate prepared as described above was incubated with 0.25 nM 1alpha ,25(OH)2[1beta -3H]D3 (specific activity, 3.4 GBq/mmol) for 15 min at 37 °C under oxygen gas in the presence or the absence of graded amounts of F2-1alpha ,25(OH)2D3. The radioactivity in the aqueous phase after extraction by the method of Bligh and Dyer (23) was measured using a scintillation counter. The lipid fraction of the reaction mixture was analyzed by reverse phase HPLC under the conditions described above after adding the chemically synthesized 1alpha ,25(OH)2D3 and 1alpha ,24R,25(OH)3D3.

Isolation and Identification of the Major Metabolite

F2-1alpha ,25(OH)2D3 (30 µg) was incubated in 150 ml of kidney homogenates obtained from rats injected with 1alpha ,25(OH)2D3 as described above. The total lipid fraction was applied to a Sep-Pak Silica column (Waters, Millford, MA). After washing the column with 40 ml/column of ethyl acetate/n-hexane (5:95) the substrate and metabolites were eluted by 50 ml of ethyl acetate/n-hexane (5:95). The eluate was concentrated and analyzed by straight phase HPLC (column, Finepak SIL 4.6 × 250 mm; mobile phase, n-hexane/chloroform/methanol, 10:3:2; flow rate, 1 ml/min) and separated in 30-s fractions. The fraction containing the major metabolite was further purified by reverse phase HPLC (column, YMC-Pack ODS-AM 4.6 × 150 mm; mobile phase, 40% acetonitrile/water; flow rate, 1 ml/min) to give 1.7 µg of a homogeneous metabolite (Fig. 1, peak X) based on the absorption at 265 nm.


Fig. 1. Effect of treating rats with 1alpha ,25(OH)2D3 on the metabolism of F2-1alpha ,25(OH)2D3 in kidney homogenates. 1 µg of 1alpha ,25(OH)2D3 or vehicle (ethanol) was injected intravenously 8 h prior to removing the kidneys. F2-1alpha ,25(OH)2[1beta -3H]D3 was incubated with kidney homogenates from vehicle-treated (upper panel) and 1alpha ,25(OH)2D3-treated rats (lower panel). The total lipid fraction of the reaction mixture was analyzed by straight phase HPLC. The peaks for the substrate F2-1alpha ,25(OH)2D3 and peak X are indicated. See "Experimental Procedures" for details.
[View Larger Version of this Image (17K GIF file)]

Mass spectra were recorded on a JEOL AX505 frit-FAB LC/MS spectrometer (Tokyo, Japan) equipped with a JASCO 880-PU HPLC system (Tokyo, Japan) (HPLC column, Develosil ODS-HG-5, 2.0 × 150 mm (Nomura Chemical Co. Ltd., Aichi, Japan)) and operated at an accelerating voltage of 3 kV in a positive ion mode. A portion of the isolated metabolite (Fig. 1, peak X, about 500 ng) was analyzed by frit-FAB LC/MS using 45% acetonitrile/water containing 0.7% glycerol matrix as the mobile phase. An aliquot of the same metabolite (500 ng) was oxidized with periodate as follows: 5% NaIO4 (20 µl) was added to a solution of the metabolite in ethanol (25 µl), and the mixture was left at room temperature for 1 h (24). The mixture was extracted with methylene chloride and analyzed by frit-FAB LC/MS using a mobile phase of 65% acetonitrile/water containing 0.7% glycerol.

Metabolism of F2-1alpha ,25(OH)2D3 in CYP24- and CYP27-transfected ROS 17/2.8 Cells

ROS 17/2.8 cells were subcultured 16-20 h prior to transfection and seeded in 100-mm plates at a density of 1 × 106 cells/dish. Cells were transfected with pSVL-CYP24, pSVL-CYP27, and pSVL-CYP27(-) according to the lipofection protocol using Lipofectin (Life Technologies, Inc.) as described in the manufacturer's manual. 24 h after transfection, the medium was replaced, and the cells were cultured for a further 24 h. Then the cells were incubated with 10 nM F2-1alpha ,25(OH)2[1beta -3H]D3 (70,000 dpm/8 ml/dish), and then methanol was added to terminate the reaction. An aliquot of lipid-soluble metabolites extracted from the cells, and their culture medium was applied to HPLC under the following conditions: column, Finepak SIL 4.6 × 250 mm; mobile phase, n-hexane/chloroform/methanol (10:2.5:1.5); flow rate, 1 ml/min; and column, J' sphere ODS-AM 4.6 × 150 mm; mobile phase, a linear gradient of acetonitrile concentration (40 to 100% in 30 min); flow rate, 1 ml/min. The radioactivity of the eluate was counted. Generation of F2-1alpha ,25,26(OH)3[1beta -3H]D3 was confirmed by comigration of the radioactivity and chemically synthesized F2-1alpha ,25,26(OH)3D3.

Northern Blots

RNA was prepared and Northern blotted as described (7). Total RNA was extracted with acid guanidine thiocyanate/phenol/chloroform. Poly(A)+ RNA was isolated by a batch method using Oligotex-dT30 super (Takara Biochemicals, Shiga, Japan). Samples (0.8 µg) of poly(A)+ RNA were resolved by electrophoresis, transferred onto a Hybond-N membrane (Amersham International plc), and probed using [alpha -32P]dCTP-labeled cDNAs for CYP24, CYP27, and beta -tubulin obtained by a random primer extension system.


RESULTS

Metabolism of F2-1alpha ,25(OH)2D3 in Rat Kidney Homogenates

No appreciable metabolism of F2-1alpha ,25(OH)2[1beta -3H]D3 proceeded after incubation with the homogenates of kidneys obtained from rats injected with the vehicle (Fig. 1, top). On the contrary, a lipid-soluble metabolite (Fig. 1, peak X) was detected in kidney homogenates from rats given 1alpha ,25(OH)2D3 (Fig. 1, bottom). The HPLC profile of peak X indicated that it was a more polar metabolite than F2-1alpha ,25(OH)2D3.

Experiments to assess the kinetics of lowering the substrate in rat kidney homogenates indicated that the Km value for F2-1alpha ,25(OH)2D3 was 2.0 µM, which was similar to that for 1alpha ,25(OH)2D3 (1.7 µM). On the other hand, the Vmax for the former was 75 pmol/min/g tissue, whereas that for the latter was 510 pmol/min/g tissue.

Purification and Identification of the Major Metabolite (Peak X) of F2-1alpha ,25(OH)2D3

To isolate the major metabolite (peak X), 30 µg of F2-1alpha ,25(OH)2D3 was incubated with 150 ml of 10% (w/v) kidney homogenates obtained from rats given 1alpha ,25(OH)2D3. The lipid extract of the metabolite was purified on a Sep-pak cartridge followed by two HPLC separations (straight and reverse phase) to yield 1.7 µg of the homogeneous metabolite (peak X). The UV spectrum of peak X was typical of the vitamin D triene system (lambda max at 265 nm, lambda min at 228 nm in ethanol). The frit-FAB LC mass spectrum (Fig. 2A) showed a parent ion at m/z 469 (M + H)+ and fragment ions at m/z 451 (M + H - H2O)+, m/z 433 (M + H - 2H2O)+, 315 (upper half fragment - H)+, and m/z 135 (bottom half fragment +H - H2O)+. This spectrum indicates that an oxygen atom was introduced to the side chain of the substrate F2-1alpha ,25(OH)2D3. To examine whether a vicinal diol function is present in the metabolite, peak X was treated with NaIO4, and the product was analyzed by frit-FAB LC/MS. HPLC showed a single major peak, and the MS spectrum of the peak showed a parent ion at 437 (M + H)+ (relative intensity, 11.5%) and fragment ions at m/z 419 (M + - H2O)+ (relative intensity, 13.3%), and m/z 401 (M + H - 2H2O)+ (relative intensity; 10.2%). These results indicate that a formyl group was removed by NaIO4 oxidation and that there is a terminal vicinal diol function in the side chain of peak X. Thus, we identified peak X as one of the 25-epimers of F2-1alpha ,25,26(OH)3D3. Finally the structure of peak X was confirmed by direct comparison with two chemically synthesized 25-epimers of F2-1alpha ,25,26(OH)3D3 by HPLC and frit-FAB LC/MS. The metabolite comigrated with the two synthesized compounds (epimers were not separated under the HPLC conditions) (Fig. 3), and the mass spectra of the isolated and synthetic epimers were identical (Fig. 2).


Fig. 2. Mass spectra of the major metabolite of F2-1alpha ,25(OH)2D3 (A) and the two chemically synthesized 25-epimers of F2-1alpha ,25,26(OH)3D3 (B and C).
[View Larger Version of this Image (33K GIF file)]


Fig. 3. Co-chromatography of the chemically synthesized F2-1alpha ,25,26(OH)3D3 epimers and the lipid-soluble fraction of the reaction mixture of kidney homogenates and F2-1alpha ,25(OH)2[1beta -3H]D3. A, UV absorption at 265 nm. B, radioactivity. Elution profiles of a mixture of the lipid fraction and synthetic standards on straight phase HPLC (column, Finepak SIL (4.6 × 250 mm); mobile phase, n-hexane/chloroform/methanol, 10:2.5:1.5; flow rate, 1 ml/min). C, UV absorption at 265 nm. D, radioactivity. Elution profiles of the mixture of the lipid fraction and synthetic standards on reverse phase HPLC (column, J' sphere ODS-AM (4.6 × 150 mm); mobile phase, a linear gradient of acetonitrile concentration (40 to 100% in 30 min); flow rate, 1 ml/min). Radioactivity corresponding to peak X comigrated with F2-1alpha ,25,26(OH)3D3 on both HPLC systems.
[View Larger Version of this Image (25K GIF file)]

Effects of F2-1alpha ,25(OH)2D3 on the Metabolism of 1alpha ,25(OH)2D3 by Renal Homogenates

The results shown in Fig. 1 suggested that 1alpha ,25(OH)2D3 induced the enzyme that catalyzed the conversion of F2-1alpha ,25(OH)2D3 into peak X (F2-1alpha ,25,26(OH)3D3). Until now, no enzymes other than CYP24 involved in vitamin D metabolism are known to be induced by 1alpha ,25(OH)2D3. CYP24 catalyzes the following series of reactions: 1alpha ,25(OH)2D3 right-arrow 1alpha ,24R,25(OH)3D3 right-arrow 24-oxo-1alpha ,25dihydroxyvitamin D3 (24-oxo-1alpha ,25(OH)2D3) right-arrow 24-oxo-1alpha ,23S,25-trihydroxyvitamin D3 (24-oxo-1alpha ,23S,25(OH)3D3) (25). The side chain of 24-oxo-1alpha ,23S,25(OH)3D3 is then cleaved and oxidized to yield water-soluble calcitroic acid (5). Therefore we examined the ability of F2-1alpha ,25(OH)2D3 to compete for the metabolism of 1alpha ,25(OH)2D3 in rat kidney homogenates. F2-1alpha ,25(OH)2D3 inhibited the conversion of 1alpha ,25(OH)2D3 into water-soluble metabolite(s) in a concentration-dependent manner (Fig. 4, left). Furthermore, HPLC analysis of the lipid-soluble fraction revealed that F2-1alpha ,25(OH)2D3 inhibited the 24-hydroxylation of 1alpha ,25(OH)2D3 (Fig. 4, right). These results indicated that F2-1alpha ,25(OH)2D3 competes with the interaction of CYP24 and 1alpha ,25(OH)2D3.


Fig. 4. Effect of F2-1alpha ,25(OH)2D3 on the metabolism of 1alpha ,25(OH)2[1beta -3H]D3 in rat kidney homogenates. 1alpha ,25(OH)2[1beta -3H]D3 was incubated with rat kidney homogenates in the presence of increasing amounts of F2-1alpha ,25(OH)2D3. Left, the yield of radioactivity in lipid-soluble (bullet ) and water-soluble fractions (open circle ) is indicated. Right, the lipid-soluble fraction was analyzed on the reverse phase HPLC (column, J' sphere ODS-AM (4.6 × 150 mm); mobile phase, a linear gradient of acetonitrile concentration (40 to 100% in 30 min); flow rate, 1 ml/min) after adding authentic 1alpha ,25(OH)2D3 and 1alpha ,24R,25(OH)3D3. The radioactivity that comigrated with 1alpha ,25(OH)2D3 (open circle ), 1alpha ,24R,25(OH)3D3 (black-triangle) and the sum of the radioactivity of other fractions (square ) are indicated. The results are expressed as the means ± S.D. of three experiments.
[View Larger Version of this Image (14K GIF file)]

Metabolism of F2-1alpha ,25(OH)2D3 in the CYP-24-transfected ROS 17/2.8 Cells

To confirm that CYP24 can catalyze the hydroxylation of F2-1alpha ,25(OH)2D3 at C-26, we examined the metabolism of F2-1alpha ,25(OH)2D3 in ROS17/2.8 cells transfected with the plasmid expressing CYP24. Expression of CYP24 and CYP27 was confirmed by Northern blotting (Fig. 5D). Control ROS17/2.8 cells expressed neither CYP24 mRNA (Fig. 5D) nor CYP24 activity (data not shown). Incubation of F2-1alpha ,25(OH)2[1beta -3H]D3 for 96 h with ROS17/2.8 cells transfected with pSVL-CYP24 generated a metabolite that comigrated with chemically synthesized F2-1alpha ,25,26(OH)3D3 (Fig. 5A). The metabolite shown as a solid peak in Fig. 5A also comigrated with the synthetic F2-1alpha ,25,26(OH)3D3 on the straight phase HPLC (column, Finepak SIL 4.6 × 250 mm; mobile phase, n-hexane/chloroform/methanol, 10:2.5:1.5; flow rate, 1 ml/min) (data not included). In contrast, F2-1alpha ,25,26(OH)3D3 was not generated in the cells transfected with pSVL-CYP27 or pSVL-CYP27(-) under the same conditions (Fig. 5, B and C). These results showed that CYP24 catalyzes the 26-hydroxylation of F2-1alpha ,25(OH)2D3.


Fig. 5. Metabolism of F2-1alpha ,25(OH)2[1beta -3H]D3 in ROS17/2.8 cells transfected with the expression plasmids for CYP24 and CYP27. A-C, metabolism of F2-1alpha ,25(OH)2[1beta -3H]D3 in cells transfected with pSVL-CYP24 (A), pSVL-CYP27 (B), and pSVL-CYP27(-) (C). The retention times of F2-1alpha ,25(OH)2D3 and F2-1alpha ,25,26(OH)3D3 are indicated. F2-1alpha ,25(OH)2D3 into F2-1alpha ,25,26(OH)3D3 (solid peak) was metabolized only in the cells transfected with pSVL-CYP24 (A). D, expression of mRNAs for exogenous CYP24 (CYP-24), exogenous CYP27 (CYP-27), and endogenous beta -tubulin (Tub) in cells transfected with pSVL-CYP24 (lane a), pSVL-CYP27 (lane b), and pSVL-CYP27(-) (lane c). The expression was also examined in the cells transfected with an empty vector (line d). The cells transfected with pSVL-CYP27(-) expressed RNA with a poly(A)+ tail that hybridized with the antisense strand of CYP27 cDNA. Neither endogenous CYP24 nor CYP27 mRNA was detected in this cell line. See "Experimental Procedures" for details.
[View Larger Version of this Image (31K GIF file)]


DISCUSSION

CYP24 was discovered as the enzyme responsible for the hydroxylation at C-24 in the metabolism of 1alpha ,25(OH)2D3 and 25(OH)D3 (25). Recently, it was found that human recombinant CYP24 also catalyzes the C-23 hydroxylation of 25(OH)D3 (11), indicating that this enzyme has multicatalytic functions. However, there is no evidence that CYP24 hydroxylates any other carbons than C-24 or C-23 of vitamin D compounds. In this study, we showed that F2-1alpha ,25(OH)2D3, C-24 of which is protected from the hydroxylation by fluorination, is metabolized into F2-1alpha ,25,26(OH)3D3 by a 1alpha ,25(OH)2D3-induced enzyme in the rat kidney (Figs. 1, 2, 3). The enzyme involved in this hydroxylation was CYP24 (Figs. 4 and 5A). This is the first report to describe that CYP24 hydroxylates a carbon other than C-24 and C-23 of vitamin D compounds. It is generally accepted that the fluorine atom mimics the hydrogen atom. A computer analysis confirmed that 24,24-F2-1alpha ,25(OH)2D3 was very similar to that of 1alpha ,25(OH)2D3, though the electronegativity and hydrophobicity of the fluorine atom were stronger than those of the hydrogen atom (data not shown). Thus the possibility cannot be ruled out at present that the fluorine atoms at C-24 influence the susceptibility of the neighboring carbons to CYP24. Hydroxylation at C-26 of F2-1alpha ,25(OH)2D3 made C-25 asymmetric. The stereochemical configuration at C-25 of this metabolite has yet to be determined.

According to the model proposed by Dilworth et al., CYP24 selects its hydroxylation site(s) by the distance from the vitamin D ring structure (13). The results of the present study, however, suggest that the hydroxylation site selected by the enzyme is not necessarily strict and that CYP24 can hydroxylate a carbon other than C-24 and C-23 when C-24 is protected from metabolic hydroxylation. Therefore, it is highly likely that CYP24 is also responsible for the C-26 hydroxylation of the vitamin D3 metabolites in vivo. At present, the possibility cannot be excluded that enzymes other than CYP24, such as CYP27, hydroxylate C-26 of 1alpha ,25(OH)2D3 in vivo. In fact, CYP27 reportedly hydroxylates C-25 and C-26 of vitamin D3 and 1alpha (OH)D3 (12). Under our conditions, however, kidney homogenates obtained from rats given either 1alpha ,25(OH)2D3 or vehicle did not metabolize vitamin D3 into 25(OH)D3 or other metabolites (data not shown), indicating that no CYP27 is present in the kidney. In addition, CYP27-transfected ROS17/2.8 cells did not metabolize F2-1alpha ,25(OH)2D3 into F2-1alpha ,25,26(OH)3D3 (Fig. 5B). Therefore, the C-26 hydroxylation of F2-1alpha ,25(OH)2D3 in kidney homogenates does not appear to be mediated by CYP27.

Two methyl groups at C-25 of 1alpha ,25(OH)2D3 (or 25(OH)D3) are heterotopic. Hydroxylation of one of the methyls yields a new chiral center at C-25. Hydroxylation of the pro-S-methyl group produces 25R configuration and pro-R-methyl group 25S configuration. Two types of 26-oxygenated vitamin D3 metabolites have been found; one is the metabolites with 25S configuration such as 1alpha ,25S,26-trihydroxyvitamin D3 (26) and 25S,26-dihydroxyvitamin D3 (27), and the other is those with 25R configuration such as 25R-hydroxyvitamin D3-26,23S-lactone (28), 1alpha ,25R-dihydroxyvitamin D3-26,23S-lactone (29), and their precursors. It has also been reported that natural 25,26-dihydroxyvitamin D3 is a mixture of 25R- and 25S-isomers (30). These results suggest that there are two C-26 hydroxylation enzymes; one catalyzes the hydroxylation of the pro-S-methyl and the other catalyzes the pro-R-methyl. It may be likely that these two types of hydroxylation at C-26 are catalyzed by CYP24 and CYP27, respectively. The stereochemical configuration at C-25 of the metabolite X, F2-1alpha ,25,26(OH)3D3, is now under investigation.

CYP24 has been found in all the target tissues of vitamin D that possess VDR (31). Cloning the cDNA and characterizing the CYP24 gene (20, 32) has allowed the mechanism of regulation of its gene expression to be studied. Northern blotting has revealed that the expression of this enzyme is induced exclusively by 1alpha ,25(OH)2D3 at the transcriptional level (6-8). Three groups independently identified functional but different vitamin D-responsive elements (VDRE-1 and VDRE-2) in the antisense strand in rat CYP24 gene promoter at -151 to -137 (VDRE-1) (33, 34) and at -259 to -245 (VDRE-2) (35) in rats. The presence of the two VDREs in the CYP24 gene promoter may be important for regulating intracellular concentration as well as the half-life of 1alpha ,25(OH)2D3. Makin et al. reported that the target cells of vitamin D metabolize 1alpha ,25(OH)2D3 sequentially into calcitroic acid by the 24-oxidation pathway (5). Using a bacterially expressed enzyme, Akiyoshi-Shibata et al. showed that CYP24 alone can catalyze all of the following reactions; 1alpha ,25(OH)2D3 right-arrow 1alpha ,24R,25(OH)3D3 right-arrow 24-oxo-1alpha ,25(OH)2D3 right-arrow 24-oxo-1alpha ,23S,25(OH)3D3 (25). The ability of CYP24 to catalyze not only 24-hydroxylation but also its successive reactions implies that the role of this enzyme is to decrease the binding affinity of vitamin D compounds to VDR in the target cells, because 1alpha ,24R,25(OH)3D3 still has about 40% of the affinity of 1alpha ,25(OH)2D3 for VDR (36). Although the bacterially expressed CYP24 also catalyzed the sequential metabolism of 25(OH)D3, namely 25(OH)D3 right-arrow 24R,25-dihydroxyvitamin D3 right-arrow 24-oxo-25-hydroxyvitamin D3 right-arrow 24-oxo-23S,25-dihydroxyvitamin D3, the Km value of the enzyme for 1alpha ,25(OH)2D3 was one-tenth of that for 25(OH)D3 (25), suggesting that the former is the real substrate of CYP24.

In conclusion, CYP24 appears to solely regulate the intracellular concentration of the VDR ligand and hence the VDR-mediated transactivation in the target cells of vitamin D. It is highly likely that CYP24 catalyzes all three known catabolic pathways of 1alpha ,25(OH)2D3, namely the C-23, C-24, and C-26 hydroxylation pathways, further emphasizing the importance of this enzyme in regulating vitamin D metabolism and function.


FOOTNOTES

*   This work was supported in part by Grants-in-Aid 08457494 and 06454527 from the Ministry of Science, Education and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger Dagger    To whom correspondence should be addressed: Tel.: 81-3-3784-8163; Fax: 81-3-3784-5555.
1   The abbreviations used are: 1alpha ,25(OH)2D3, 1alpha ,25-dihydroxyvitamin D3; 25(OH)D3, 25-hydroxyvitamin D3; 23S,25(OH)2D3, 23S,25-dihydroxyvitamin D3; 1alpha (OH)D3, 1alpha -hydroxyvitamin D3; 1alpha ,24R,25(OH)3D3, 1alpha ,24R,25-trihydroxyvitamin D3; 24-oxo-1alpha ,25(OH)2D3, 24-oxo-1alpha ,25-dihydroxyvitamin D3; 24-oxo-1alpha ,23S,25(OH)3D3, 24-oxo-1alpha ,23S,25trihydroxyvitamin D3; F2-1alpha ,25(OH)2-D3, 24,24-difluoro-1alpha ,25-dihydroxy-vitamin D3; F2-1alpha ,25,26(OH)3D3, 24,24-difluoro-1alpha ,25,26-trihydroxy-vitamin D3; 1alpha ,25(OH)2[1beta -3H]D3, 1alpha ,25-dihydroxy[1beta -3H]vitamin D3; F2-1alpha ,25-(OH)2[1beta -3H]D3, F2-1alpha ,25-dihydroxy[1beta -3H]vitamin D3; HPLC, high pressure liquid chromatography; FAB, fast atom bombardment; LC, liquid chromatography; MS, mass spectroscopy; VDR, vitamin D receptor; VDRE, vitamin D-responsive element.
2   Y. Miyamoto, T. Shinki, K. Yamamoto, Y. Ohyama, H. Iwasaki, R. Hosotani, T. Kasama, H. Takayama, S. Yamada, and T. Suda, unpublished results.

REFERENCES

  1. Ishizuka, S., Kiyoki, M., Orimo, H., and Norman, A. W. (1985) in Vitamin D: Chemical, Biochemical and Clinical Update (Norman, A. W., Scheafer, K., Grigoleit, H.-G., and Harrath, D. v., eds), pp. 402-403, Walter de Gruyter & Co., Berlin, New York
  2. Mayer, E., Bishop, J. E., Chandraratna, R. A. S., Okamura, W. H., Kruse, J. R., Popjak, G., Ohmura, N., and Norman, A. W. (1983) J. Biol. Chem. 258, 13458-13465 [Abstract/Free Full Text]
  3. Reddy, G. S., Tserng, K.-Y., Thoma, B. R., Dayal, R., and Norman, A. W. (1987) Biochemistry 26, 324-331 [Medline] [Order article via Infotrieve]
  4. Tanaka, Y., Schnoes, H. K., Smith, C. M., and DeLuca, H. F. (1981) Arch. Biochem. Biophys. 210, 104-109 [Medline] [Order article via Infotrieve]
  5. Makin, G., Lohnes, D., Byford, V., Ray, R., and Jones, G. (1989) Biochem. J. 262, 173-180 [Medline] [Order article via Infotrieve]
  6. Armbrecht, H. J., and Boltz, M. A. (1991) FEBS Lett. 292, 17-20 [CrossRef][Medline] [Order article via Infotrieve]
  7. Shinki, T., Jin, C. H., Nishimura, A., Nagai, Y., Ohyama, Y., Noshiro, M., Okuda, K., and Suda, T. (1992) J. Biol. Chem. 267, 13757-13762 [Abstract/Free Full Text]
  8. Nishimura, A., Shinki, T., Jin, C. H., Ohyama, Y., Noshiro, M., Okuda, K., and Suda, T. (1994) Endocrinology 134, 1794-1799 [Abstract]
  9. Suda, T., Shinki, T., and Kurokawa, K. (1994) Curr. Opin. Nephrol. Hypertens. 3, 59-64 [Medline] [Order article via Infotrieve]
  10. Siu-Caldera, M.-L., Zou, L., Ehrlich, M. G., Schwartz, E. R., Ishizuka, S., and Reddy, G. S. (1995) Endocrinology 136, 4195-4203 [Abstract]
  11. Beckman, M. J., Tadikonda, P., Werner, E., Prahl, J., Yamada, S., and DeLuca, H. F. (1996) Biochemistry 35, 8465-8472 [CrossRef][Medline] [Order article via Infotrieve]
  12. Guo, Y.-D., Strugnell, S., Back, D. W., and Jones, G. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8668-8672 [Abstract/Free Full Text]
  13. Dilworth, F. J., Scott, I., Green, A., Strugnell, S., Guo, Y.-D., Roberts, E. A., Kremer, R., Calverley, M. J., Makin, H. L. J., and Jones, G. (1995) J. Biol. Chem. 270, 16766-16774 [Abstract/Free Full Text]
  14. Yamada, S., Ohmori, M., and Takayama, H. (1979) Chem. & Pharm. Bull. (Tokyo) 27, 3196-3198
  15. Kobakoff, B. D., Kendrick, N. C., Faber, D., and DeLuca, H. F. (1982) Arch. Biochem. Biophys. 215, 582-588 [Medline] [Order article via Infotrieve]
  16. Shiina, Y., Abe, E., Miyaura, C., Tanaka, H., Yamada, S., Ohmori, M., Nakayama, K., Takayama, H., Matsunaga, I., Nishii, Y., DeLuca, H. F., and Suda, T. (1983) Arch. Biochem. Biophys. 220, 90-94 [Medline] [Order article via Infotrieve]
  17. Miyamoto, Y., Shinki, T., Ohyama, Y., Kasama, T., Iwasaki, H., Hosotani, R., Sato, T., and Suda, T. (1995) J. Biochem. (Tokyo) 118, 1068-1076 [Abstract]
  18. Ando, K., Kondo, F., Koike, F., and Takayama, H. (1992) Chem. & Pharm. Bull. (Tokyo) 40, 1662-1664
  19. Holick, S. A., Holick, M. F., and MacLaughlin, J. A. (1980) Biochem. Biophys. Res. Commun. 97, 1031-1037 [Medline] [Order article via Infotrieve]
  20. Ohyama, Y., Noshiro, M., and Okuda, K. (1990) FEBS Lett. 278, 195-198 [CrossRef]
  21. Usui, E., Noshiro, M., Ohyama, Y., and Okuda, K. (1990) FEBS Lett. 274, 175-177 [CrossRef][Medline] [Order article via Infotrieve]
  22. Yamada, S., Ino, E., Takayama, H., Horiuchi, N., Shinki, T., Suda, T., Jones, G., and DeLuca, H. F. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 7485-7489 [Abstract]
  23. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-917
  24. Suda, T., DeLuca, H. F., Schnoes, H. K., Tanaka, Y., and Holick, M. F. (1970) Biochemistry 9, 4776-4780 [Medline] [Order article via Infotrieve]
  25. Akiyoshi-Shibata, M., Sasaki, T., Ohyama, Y., Noshiro, M., Okuda, K., and Yabusaki, Y. (1994) Eur. J. Biochem. 224, 335-343 [Abstract]
  26. Partridge, J. J., Shiuey, S.-J., Chandha, N. K., Baggiolini, E. G., Hennessy, B. M., Uskokovic, M. R., Napoli, J. L., Reinhardt, T. A., and Horst, R. L. (1981) Helv. Chim. Acta 64, 2138-2141
  27. Partridge, J. J., Shiuey, S.-J., Chandha, N. K., Baggiolini, E. G., Blount, J. F., and Uskokovic, M. R. (1981) J. Am. Chem. Soc. 103, 1253-1255
  28. Yamada, S., Nakayama, K., Takayama, H., Shinki, T., Takasaki, Y., and Suda, T. (1984) J. Biol. Chem. 259, 884-889 [Abstract/Free Full Text]
  29. Ishizuka, S., Oshida, J., Tsuruta, H., and Norman, A. W. (1985) Arch. Biochem. Biophys. 242, 82-89 [Medline] [Order article via Infotrieve]
  30. Ikekawa, N., Noizumi, N., Ohshima, E., Ishizuka, S., Takeshita, T., Tanaka, Y, and DeLuca, H. F. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 5286-5288 [Abstract]
  31. Pike, J. W. (1991) Annu. Rev. Nutr. 11, 189-216 [CrossRef][Medline] [Order article via Infotrieve]
  32. Ohyama, Y., Noshiro, M., Eggertsen, G., Gotoh, O., Kato, Y., Björkhem, I., and Okuda, K. (1993) Biochemistry 32, 76-82 [Medline] [Order article via Infotrieve]
  33. Ohyama, Y., Ozono, K., Uchida, M., Shinki, T., Kato, S., Suda, T., Yamamoto, O., Noshiro, M., and Kato, Y. (1994) J. Biol. Chem. 269, 10545-10550 [Abstract/Free Full Text]
  34. Hahn, C. N., Kerry, D. M., Omdahl, J. L., and May, B. K. (1994) Nucleic Acids Res. 22, 2410-2416 [Abstract]
  35. Zierold, C., Darwish, H. M., and DeLuca, H. F. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 900-902 [Abstract]
  36. Bouillon, R., Okamura, W. H., and Norman, A. W. (1995) Endocr. Rev. 16, 200-257 [Medline] [Order article via Infotrieve]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.