(Received for publication, October 22, 1996, and in revised form, March 17, 1997)
From the The sequential oxidation and cleavage of the side
chain of 1 Metabolic inactivation of 1 Of numerous synthetic analogs of
1 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-1 All chemicals were of highly purified analytical
grade. F2-1 1 µg of 1 The renal homogenate prepared as described above was
incubated with 0.25 nM
1 F2-1
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.
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( 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
[ No appreciable metabolism of
F2-1 Experiments to assess the kinetics of lowering the substrate in rat
kidney homogenates indicated that the Km value for
F2-1 To isolate
the major metabolite (peak X), 30 µg of
F2-1
The results shown in Fig. 1 suggested that
1
To confirm that CYP24
can catalyze the hydroxylation of
F2-1
CYP24 was discovered as the enzyme responsible for the
hydroxylation at C-24 in the metabolism of
1 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
1 Two methyl groups at C-25 of 1 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
1 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
1
Department of Biochemistry,
Graduate Department of Gene Science,
,25-dihydroxyvitamin D3
(1
,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 1
,25(OH)2D3,
24,24-difluoro-1
,25-dihydroxyvitamin D3
(F2-1
,25(OH)2D3), C-24 of which
was designed to resist metabolic hydroxylation. When kidney homogenates
prepared from 1
,25(OH)2D3-supplemented rats
were incubated with
F2-1
,25(OH)2D3, it was mainly
converted to a more polar metabolite. We isolated and unequivocally
identified the metabolite as
24,24-difluoro-1
,25,26-trihydroxyvitamin D3 (F2-1
,25,26(OH)3D3) by
ultraviolet absorption spectrometry, frit-fast atom bombardment liquid
chromatography/mass spectroscopy analysis, and direct comparison with
chemically synthesized
F2-1
,25,26(OH)3D3. Metabolism of F2-1
,25(OH)2D3
into F2-1
,25,26(OH)3D3 by kidney homogenates was induced by the prior administration of
1
,25(OH)2D3 into rats. The C-24 oxidation of
1
,25(OH)2D3 in renal homogenates was
inhibited by F2-1
,25(OH)2D3 in a
concentration-dependent manner. Moreover,
F2-1
,25,26(OH)3D3 was formed in
ROS17/2.8 cells transfected with a plasmid expressing
1
,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
1
,25(OH)2D3 but also at C-26 of
F2-1
,25(OH)2D3, indicating that
this enzyme has a broader substrate specificity of the hydroxylation
sites than previously considered.
,25-dihydroxyvitamin D3
(1
,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
1
,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
1
,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 1
,25(OH)2D3 initiated by
C-23 hydroxylation is induced by 1
,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 1
,25(OH)2D3. In
contrast, mitochondrial vitamin D-25-hydroxylase (CYP27)
catalyzes the hydroxylation at C-25 and C-26 of vitamin D3
and 1
-hydroxyvitamin D3 (1
(OH)D3) (12),
but it is not clear whether or not this enzyme hydroxylates C-26 of
1
,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).
,25(OH)2D3,
24,24-difluoro-1
,25-dihydroxyvitamin D3
(F2-1
,25(OH)2D3) (14) was the
first that had a higher biological activity than the parental
1
,25(OH)2D3 (15-17). Although the
biological activity of
F2-1
,25(OH)2D3 was higher, the
binding affinity of this analog to VDR was almost identical to that of
1
,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
F2-1
,25(OH)2D3, however, has not
yet been clarified. We recently reported that
F2-1
,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,
F2-1
,25(OH)2D3 itself (17).
,25,26-trihydroxyvitamin D3
(F2-1
,25,26(OH)3D3) as a major
metabolite of F2-1
,25(OH)2D3.
Moreover, the enzyme catalyzing the conversion of
F2-1
,25(OH)2D3 into
F2-1
,25,26(OH)3D3 was CYP24.
Materials
,25(OH)2D3 (18),
(25R)- and
(25S)-F2-1
,25,26(OH)3D32
were synthesized in our laboratory. The
1
,25-dihydroxy[1
-3H]vitamin D3
(1
,25(OH)2[1
-3H]D3)
(specific activity, 202 GBq/mmol) and
24,24-difluoro-1
,25-dihydroxy[1
-3H]vitamin
D3
(F2-1
,25(OH)2[1
-3H]D3)
(specific activity, 396 GBq/mmol) were prepared by Daiichi Pure
Chemicals, Co., Ltd. (Tokyo, Japan) as described (19). 1
,25(OH)2D3 was purchased from Wako Pure
Chemicals (Osaka, Japan). 1
,24R,25-trihydroxyvitamin
D3 (1
,24R,25(OH)3D3)
was a gift from Kureha Chemical Industries (Tokyo, Japan).
[
-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
-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).
,25(OH)2D3 by Rat Renal
Homogenates
,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
F2-1
,25(OH)2[1
-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).
,25(OH)2D3 by Rat Renal
Homogenates
,25(OH)2[1
-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 F2-1
,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 1
,25(OH)2D3 and
1
,24R,25(OH)3D3.
,25(OH)2D3
(30 µg) was incubated in 150 ml of kidney homogenates obtained from
rats injected with 1
,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
1,25(OH)2D3 on the metabolism of
F2-1
,25(OH)2D3 in kidney
homogenates. 1 µg of 1
,25(OH)2D3 or
vehicle (ethanol) was injected intravenously 8 h prior to removing the kidneys.
F2-1
,25(OH)2[1
-3H]D3
was incubated with kidney homogenates from vehicle-treated (upper
panel) and 1
,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 F2-1
,25(OH)2D3 and
peak X are indicated. See "Experimental Procedures" for
details.
[View Larger Version of this Image (17K GIF file)]
,25(OH)2D3 in CYP24- and
CYP27-transfected ROS 17/2.8 Cells
) 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
F2-1
,25(OH)2[1
-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
F2-1
,25,26(OH)3[1
-3H]D3
was confirmed by comigration of the radioactivity and chemically synthesized
F2-1
,25,26(OH)3D3.
-32P]dCTP-labeled cDNAs for CYP24, CYP27, and
-tubulin obtained by a random primer extension system.
Metabolism of F2-1,25(OH)2D3
in Rat Kidney Homogenates
,25(OH)2[1
-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
1
,25(OH)2D3 (Fig. 1, bottom). The HPLC profile of peak X indicated that it was a more polar metabolite than F2-1
,25(OH)2D3.
,25(OH)2D3 was 2.0 µM, which was similar to that for 1
,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.
,25(OH)2D3
,25(OH)2D3 was incubated
with 150 ml of 10% (w/v) kidney homogenates obtained from rats given
1
,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 (
max at 265 nm,
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 F2-1
,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 + H
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 F2-1
,25,26(OH)3D3.
Finally the structure of peak X was confirmed by direct comparison with two chemically synthesized 25-epimers of
F2-1
,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
F2-1,25(OH)2D3 (A)
and the two chemically synthesized 25-epimers of
F2-1
,25,26(OH)3D3 (B
and C).
[View Larger Version of this Image (33K GIF file)]
Fig. 3.
Co-chromatography of the chemically
synthesized F2-1,25,26(OH)3D3
epimers and the lipid-soluble fraction of the reaction mixture of
kidney homogenates and
F2-1
,25(OH)2[1
-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 F2-1
,25,26(OH)3D3 on both
HPLC systems.
[View Larger Version of this Image (25K GIF file)]
,25(OH)2D3 on
the Metabolism of 1
,25(OH)2D3 by Renal
Homogenates
,25(OH)2D3 induced the enzyme that
catalyzed the conversion of
F2-1
,25(OH)2D3 into peak X
(F2-1
,25,26(OH)3D3). Until now,
no enzymes other than CYP24 involved in vitamin D metabolism are known
to be induced by 1
,25(OH)2D3. CYP24
catalyzes the following series of reactions: 1
,25(OH)2D3
1
,24R,25(OH)3D3
24-oxo-1
,25dihydroxyvitamin D3
(24-oxo-1
,25(OH)2D3)
24-oxo-1
,23S,25-trihydroxyvitamin D3
(24-oxo-1
,23S,25(OH)3D3) (25).
The side chain of
24-oxo-1
,23S,25(OH)3D3 is then
cleaved and oxidized to yield water-soluble calcitroic acid (5).
Therefore we examined the ability of
F2-1
,25(OH)2D3 to compete for
the metabolism of 1
,25(OH)2D3 in rat kidney
homogenates. F2-1
,25(OH)2D3
inhibited the conversion of 1
,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
F2-1
,25(OH)2D3 inhibited the
24-hydroxylation of 1
,25(OH)2D3 (Fig. 4,
right). These results indicated that F2-1
,25(OH)2D3 competes with the
interaction of CYP24 and 1
,25(OH)2D3.
Fig. 4.
Effect of
F2-1,25(OH)2D3 on the metabolism
of 1
,25(OH)2[1
-3H]D3 in rat
kidney homogenates.
1
,25(OH)2[1
-3H]D3 was
incubated with rat kidney homogenates in the presence of increasing
amounts of F2-1
,25(OH)2D3.
Left, the yield of radioactivity in lipid-soluble (
) and
water-soluble fractions (
) 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 1
,25(OH)2D3 and 1
,24R,25(OH)3D3. The
radioactivity that comigrated with
1
,25(OH)2D3 (
),
1
,24R,25(OH)3D3 (
) and the sum
of the radioactivity of other fractions (
) are indicated. The
results are expressed as the means ± S.D. of three
experiments.
[View Larger Version of this Image (14K GIF file)]
,25(OH)2D3
in the CYP-24-transfected ROS 17/2.8 Cells
,25(OH)2D3 at C-26, we
examined the metabolism of
F2-1
,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
F2-1
,25(OH)2[1
-3H]D3
for 96 h with ROS17/2.8 cells transfected with pSVL-CYP24 generated a metabolite that comigrated with chemically synthesized F2-1
,25,26(OH)3D3 (Fig.
5A). The metabolite shown as a solid peak in Fig.
5A also comigrated with the synthetic
F2-1
,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,
F2-1
,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
F2-1
,25(OH)2D3.
Fig. 5.
Metabolism of
F2-1,25(OH)2[1
-3H]D3
in ROS17/2.8 cells transfected with the expression plasmids for CYP24
and CYP27. A-C, metabolism of
F2-1
,25(OH)2[1
-3H]D3
in cells transfected with pSVL-CYP24 (A), pSVL-CYP27
(B), and pSVL-CYP27(
) (C). The retention times
of F2-1
,25(OH)2D3 and
F2-1
,25,26(OH)3D3 are indicated.
F2-1
,25(OH)2D3 into
F2-1
,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
-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)]
,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
F2-1
,25(OH)2D3, C-24 of which is
protected from the hydroxylation by fluorination, is metabolized into
F2-1
,25,26(OH)3D3 by a
1
,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-F2-1
,25(OH)2D3 was very
similar to that of 1
,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 F2-1
,25(OH)2D3 made C-25
asymmetric. The stereochemical configuration at C-25 of this metabolite
has yet to be determined.
,25(OH)2D3 in vivo. In fact,
CYP27 reportedly hydroxylates C-25 and C-26 of vitamin D3
and 1
(OH)D3 (12). Under our conditions, however, kidney
homogenates obtained from rats given either
1
,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
F2-1
,25(OH)2D3 into
F2-1
,25,26(OH)3D3 (Fig. 5B). Therefore, the C-26 hydroxylation of
F2-1
,25(OH)2D3 in kidney homogenates does not appear to be mediated by CYP27.
,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
1
,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), 1
,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,
F2-1
,25,26(OH)3D3, is now under
investigation.
,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
1
,25(OH)2D3. Makin et al.
reported that the target cells of vitamin D metabolize 1
,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;
1
,25(OH)2D3
1
,24R,25(OH)3D3
24-oxo-1
,25(OH)2D3
24-oxo-1
,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
1
,24R,25(OH)3D3 still has about
40% of the affinity of 1
,25(OH)2D3 for VDR
(36). Although the bacterially expressed CYP24 also catalyzed the
sequential metabolism of 25(OH)D3, namely
25(OH)D3
24R,25-dihydroxyvitamin D3
24-oxo-25-hydroxyvitamin D3
24-oxo-23S,25-dihydroxyvitamin D3, the
Km value of the enzyme for
1
,25(OH)2D3 was one-tenth of that for
25(OH)D3 (25), suggesting that the former is the real
substrate of CYP24.
,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.
*
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.
To whom correspondence should be addressed: Tel.:
81-3-3784-8163; Fax: 81-3-3784-5555.
1
The abbreviations used are:
1,25(OH)2D3, 1
,25-dihydroxyvitamin
D3; 25(OH)D3, 25-hydroxyvitamin
D3; 23S,25(OH)2D3,
23S,25-dihydroxyvitamin D3;
1
(OH)D3, 1
-hydroxyvitamin D3;
1
,24R,25(OH)3D3,
1
,24R,25-trihydroxyvitamin D3;
24-oxo-1
,25(OH)2D3,
24-oxo-1
,25-dihydroxyvitamin D3;
24-oxo-1
,23S,25(OH)3D3, 24-oxo-1
,23S,25trihydroxyvitamin D3;
F2-1
,25(OH)2-D3,
24,24-difluoro-1
,25-dihydroxy-vitamin D3;
F2-1
,25,26(OH)3D3,
24,24-difluoro-1
,25,26-trihydroxy-vitamin D3;
1
,25(OH)2[1
-3H]D3,
1
,25-dihydroxy[1
-3H]vitamin D3;
F2-1
,25-(OH)2[1
-3H]D3,
F2-1
,25-dihydroxy[1
-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.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.