Centre de recherche, Hôpital Saint-Luc, Centre Hospitalier de l'Université de Montréal, Département de pharmacologie, Faculté de médecine, Université de Montréal, Montreal, Quebec, Canada H2X 1P1
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ABSTRACT |
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The vitamin D3-25-hydroxylase CYP27A is located predominantly in liver, but its expression is also detected in extrahepatic tissues. Our aim was to evaluate the regulation of CYP27A by vitamin D3 (D3) or its metabolites in rat duodena. Vitamin D-depleted rats were repleted with D3, 25-hydroxyvitamin D (25OHD), or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or acutely injected 1,25(OH)2D3 to investigate the mechanisms of action of the hormone. All D3 compounds led to a progressive decrease in CYP27A mRNA, with levels after D3 representing 20% of that observed in D depletion. 25OHD decreased CYP27A mRNA by 55%, whereas 1,25(OH)2D3 led to a 40% decrease, which was accompanied by a 31% decrease in CYP27A protein levels and an 89% decrease in enzyme activity. Peak circulating 1,25(OH)2D3 concentrations were, however, the highest in D3-repleted, followed by 25OHD- and 1,25(OH)2D3-repleted animals. 1,25(OH)2D3 resulted in a decrease in both CYP27A mRNA half-life and transcription rate. Our data illustrate that the intestine expresses the D3-25-hydroxylase and that the gene is highly regulated in vivo through a direct action of 1,25(OH)2D3 or through the local production of D3 metabolites.
25-hydroxyvitamin D3
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INTRODUCTION |
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VITAMIN
D3 (d3) of endogenous origin and ingested
vitamin D2 (D2) [collectively referred to as
vitamin D (D)] are natural secosteroids that have, in their native
forms, no biological activity. D exhibits a short circulating half-life
and is efficiently captured by storage sites such as adipose tissues
and muscles and by the liver, where the vitamin undergoes its first
anabolic biotransformation through a C-25 hydroxylation reaction.
The hepatic product 25-hydroxyvitamin D (25OHD) is rapidly exported to
the systemic circulation, where its half-life has been reported to be
in the order of 2-3 wk in humans. This long half-life
associated with the efficient hepatic capture and hydroxylation of the
parent compound makes 25OHD the most reliable marker of D nutritional
status in humans and laboratory animals. At physiological
concentrations, 25OHD is not, however, known to have any significant
biological activity but must undergo regulated hydroxylation steps at
C-1 or C-24 in the kidney to achieve full biological action through
either its hormonal form, 1a,25-dihydroxyvitamin D3
[1,25(OH)2D3], or the now-active bone and
cartilage candidate, 24,25-dihydroxyvitamin D3
[24,25(OH)2D3] (39). Other D
compounds, such as 1
-hydroxyvitamin D3
(1
OHD3) and dihydrotachysterol, must also undergo the
necessary C-25 hydroxylation step to acquire their full biological
activity (11, 26, 41).
To date, two independent monooxygenase systems active on the C-25 hydroxylation of the D3 or D2 family of compounds have been described. Bhattacharya et al. (9) first reported the enzyme to be located in the hepatic microsomal fraction, whereas a few years later, Bjorkhem and Holmberg (10) reported that the mitochondrial sterol 27-hydroxylase exhibited enzyme activities towards the C-25 hydroxylation of D3. The molecular identity of the human or rat microsomal D3-25-hydroxylase has not yet been reported, although the mitochondrial enzyme has been cloned in several species and the gene encoding the enzyme termed CYP27A (3, 14, 26, 49, 54).
Studies on the kinetics of the hepatic D3-25-hydroxylases
have demonstrated that the affinity of the microsomal enzyme is much
higher (as indicated by a significantly lower Michaelis-Menten constant) than that of the mitochondrial enzyme, an observation that
has led to the conclusion that the microsomal
D3-25-hydroxylase is most likely physiologically more
relevant than its mitochondrial counterpart (22). The
intestine, however, is likely to be exposed to significantly higher
concentrations of D compounds than the liver. Indeed, the vitamin of
both dietary and pharmacological origins will be presented to the small
intestine, whereas only a fraction of the vitamin of either endogenous
or exogenous origin will be captured by the liver (24,
25). These observations strongly indicate that, in the small
intestine, the physiological significance of the mitochondrial
D3-25-hydroxylase may be highly relevant. Interestingly,
the small intestine has been reported to express CYP27A
(3) and to also exhibit enzyme activities related to
the hydroxylation of D compounds at C-24 (5, 36, 47),
whereas the Caco-2 cell line (a line closely resembling one in the
small intestine) (30) has been reported to be able to
hydroxylate 25OHD at C-1 (16), indicating that the
small intestine, in addition to its response to the D3
endocrine system, may be fully able to regulate its own D metabolism
and to respond to its local D-dependent needs through an auto- and/or
intracrine process.
To date, studies on the regulation of CYP27A gene products have focused solely on their significance and importance in relation to the biosynthesis of bile acids, and the molecular mechanisms by which CYP27A is regulated by D3 or its metabolites, most particularly in intestine, are presently unknown. The aims of this study were, therefore, 1) to determine the effect of an in vivo exposure to D3, 25OHD, or 1,25(OH)2D3 on the level of the CYP27A gene transcript in rat duodena and 2) to evaluate the mechanisms by which 1,25(OH)2D3 regulates its expression.
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MATERIALS AND METHODS |
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Experimental Design
The influence of the D endocrine system on the steady-state expression of the duodenal CYP27A was studied in D-depleted rats repleted with either calcium alone, D3, 25OHD, or 1,25(OH)2D3. To investigate the mechanism of action by which 1,25(OH)2D3 influenced the expression of CYP27A, studies were conducted on the half-life as well as the transcription rate of the gene. The effect of known cytochrome P-450 inducers on the steady-state levels of CYP27A mRNA was also evaluated.Animals
All animals used during the experiments were treated according to the standards of ethics for animal experimentation of the Canadian Council on Animal Care. All protocols were approved by the local animal ethics committee.Studies on the characterization of the CYP27A fragment
generated in our laboratory were done in normal male Sprague-Dawley rats (50). Hypocalcemic D-depleted male rats (DCa
)
were obtained as previously described (21, 27). Animals
were then submitted to experimental protocols aimed at achieving an in
vivo repletion with dietary calcium alone or with physiological
concentrations of D3, 25OHD, or
1,25(OH)2D3, as described in the next section.
Repletion With Calcium, D3, 25(OH)D, or 1,25(OH)2D3
Expression of the CYP27A duodenal gene transcript was first studied under steady-state conditions in D-depleted animals and in animals repleted with calcium alone or with D3 compounds. Repletion with calcium alone was achieved by an oral supplementation with a 3% calcium gluconate solution as drinking water for a period of 7 days, as previously described (21, 27). Repletion with D3 compounds was achieved by intraperitoneal implantation of miniosmotic pumps (Alza, Palo Alto, CA) containing D3 at a dose of 6.5 nmol/day, 25OHD at a dose of 28 pmol/day, or 1,25(OH)2D3 also at a dose of 28 pmol/day (21, 27). All compounds were administered in vehicle containing 95% ethanol-propylene glycol-0.9% saline, 3:13:4, vol/vol/vol. At the time of minipump implantation, a loading dose of 3.2 nmol D3, 14 pmol 25OHD, or 14 pmol 1,25(OH)2D3 was administered intraperitoneally to rapidly raise serum concentrations of D3, 25OHD, or 1,25(OH)2D3 and, hence, accelerate the establishment of steady-state conditions. D-depleted animals were implanted with miniosmotic pumps containing vehicle only. Repleted rats were given a 0.5% calcium gluconate solution as drinking water, whereas D-depleted controls received demineralized water. Animals repleted with D3 were killed after 1 wk of repletion, and animals repleted with 25OHD or 1,25(OH)2D3 were killed 1, 3, 5, or 7 days after initiation of the repletion protocol. At the time of euthanasia, the animals were between 7 and 8 wk of age.Treatment With Cytochrome P-450 Inducers
Studies on the induction of the gene encoding CYP27A were achieved using xenobiotics known to induce cytochrome P-450 izozymes. Normal male rats were exposed to dexamethasone (single ip injection: 400 mg/kg), 3-methylcholanthrene (single ip injection: 30 mg/kg),Half-Life Of The CYP27A Gene Transcript
Studies on the half-life of the CYP27A gene transcript were achieved in D-depleted rats subjected to a single intravenous dose (0, 2.4, 12, 120, or 240 nmol/kg) of 1,25(OH)2D3. Pharmacological hormonal concentrations were used to rapidly achieve the 1,25(OH)2D3 effect on the CYP27A gene before the production of significant downstream metabolites of the hormone. A single intraperitoneal dose of 0.5mg/kg of actinomycin D dissolved in 95% ethanol-saline (1:1 vol/vol) was administered 3 h after exposure to 1,25(OH)2D3, and animals were killed before and 1, 3, or 6 h after actinomycin D administration. CYP27A mRNA levels were evaluated as described in the following section.Transcription Rate Of The Gene Encoding CYP27A
The transcription rate of the gene encoding CYP27A was evaluated in duodenal nuclei obtained from either D25-Hydroxylase Activity
The mitochondrial 25-hydroxylase activity was measured in freshly isolated duodenal mitochondria obtained from D-depleted rats and from animals repleted by intraperitoneal miniosmotic pumps containing 28 pmol/day 1,25(OH)2D3 for a period of 7 days as described above. 1Duodenal mucosal cells were gently scrapped off, and mitochondria were
isolated as described by Rosenberg and Kappas (42). The
final mitochondrial pellet was resuspended in 0.25 M sucrose, 10 mM
Tris, 10 mM KCl, 1 mM EDTA, and 3 U/ml heparin, pH 7.4. Protein
concentration was determined according to Bradford (13). The incubation reaction (0.4-0.6 mg protein in 1.0 ml) contained 40 mM potassium phosphate, 0.25 M sucrose, 200 µM EDTA, 20 mM MgCl2, 0.2 mg BSA, 2 mg N,N'-diphenylphenylelenediamine
(Aldrich Chemical, Milwaukee, WI), and 10 mM isocitric acid (Sigma
Chemical, St. Louis, MO), pH 7.4. The reaction was started with 20 nmol 1OHD3 and allowed to continue for 40 min at 37°C under
gentle shaking. Blank reactions were carried out with boiled
mitochondria. The reaction was terminated with 3.75 ml of
chloroform-methanol (1:2 vol/vol), and 6,000 cpm
[3H]1,25(OH)2D3 were added to
correct for recovery during the extraction and chromatographic
procedures. Reaction mixtures were extracted twice, as described by
Bligh and Dyer (12). After extraction and evaporation, the
residue was dissolved in 150 ml of hexane and injected into a Beckman
model 160 HPLC (Beckman Instruments, Palo Alto, CA) fitted with a
Zorbax-Sil column (4.6 × 250 mm; Du Pont Instruments, Wilmington,
DE) and eluted in hexane-isopropanol (9:1 vol/vol) at a flow rate of 2 ml/min. Metabolites were detected at 254 nm. The fractions corresponded
to crystalline 1,25(OH)D3 (retention time 15 min without
overlap from 1
OHD3) were collected and counted (Beta
LS1801 spectrometer, Beckman). The identity of the product formed was
confirmed by a second HPLC on a C18 column eluted with
hexane-isopropanol (8:2 vol/vol).
Experimental Procedures
Determination of circulating Ca2+ and D metabolites. Serum Ca2+ concentrations were measured with an ICA2 ionized calcium analyzer (Radiometer, Copenhagen, Denmark). Serum 25OHD and 1,25(OH)2D3 concentrations were measured using the Incstar 25OHD and 1,25(OH)2D3 RIA assay kits (Incstar, Stillwater, MN) according to the manufacturer's instructions.
Molecular biology procedures.
At the time of euthanasia, the duodena and livers were isolated and
immediately frozen in liquid nitrogen and stored at 80°C until RNA extraction.
Nuclear run-on transcriptional assay.
Nuclei were isolated from duodena of DCa
or of
1,25(OH)2D3-repleted rats by the method of
Widnell and Tata (56) with the use of successive sucrose
gradient centrifugations. The nuclei obtained were resuspended in
storage buffer [40% glycerol, 5 mM MgCl2, 10 mM Tris, pH
7.4, 1 mM dithiothreitol (DTT), and 1 mM EDTA] and stored at
80°C.
The rate of CYP27A gene transcription was measured using a
previously described nuclear run-on transcriptional assay
(43) with the following modifications. Nuclei were
pelleted by centrifugation and resuspended in 50 µl of nuclear
run-off reaction mixture {50 mM Tris, pH 7.5, 50 mM
MgCl2, 2 mM DTT, 2 mM spermidine, 25 U RNase inhibitor, 1 mM ATP, 1 mM CTP, 1 mM GTP, and 50 µCi [32P]UTP (3,000 Ci/mmol, Amersham Pharmacia Biotech)} and incubated at 30°C for 60 min. The labeled RNA was hybridized to nylon membranes on which 300 ng
of the 404-bp D3-25-hydroxylase cDNA fragment with 150 ng
of 18S ribosomal RNA cDNA fragment (positive control) and 100 ng of pBS
(negative control) had been dotted and hybridized in 5% SDS, 400 mM
NaPO4, pH 7.2, 1 mM EDTA, 1 mg/ml BSA, 50% formamide, and
240 µg/ml of salmon sperm DNA. The membranes were prehybridized for
4 h at 52°C in hybridization solution without labeled RNA, and
then hybridization was performed at 52°C for 72 h. The membranes were washed and exposed to X-ray films for 14 days, and densitometry was performed as previously described (18, 35).
Western analyses of the CYP27A protein. The relative levels of CYP27A protein were determined by Western blot analyses. Membranes from intestinal samples were disrupted by sonication and homogenized in 100 mM Tris, pH 7.6, 3 mM phenylmethylsulfonyl fluoride, 300 mM KCl, and 1% BSA and centrifuged at 100 000 g, and the supernatant was precipitated with 40% ammonium sulfate overnight. Thirty micrograms of proteins were loaded onto an SDS-PAGE 5-15% gradient acrylamide gel and transblotted onto polyvinylidene difluoride membranes. The membranes were first incubated for 1 h with a rabbit polyclonal antibody raised against human CYP27A (1:1,000; gift from Dr. David Russel, University of Texas Southwestern Medical Center, Dallas, TX), followed by an incubation with an anti-rabbit IgG streptavidin-biotinylated species-specific antibody (1:1,000; Amersham Pharmacia Biotech), and finally incubated with a streptavidin-biotinylated horseradish-peroxidase complex (1:1,000; Amersham Pharmacia Biotech). The antigen-antibody complex was visualized with 3,3'-diaminobenzidine (Sigma Chemical, Mississauga, ON, Canada). Quantification was achieved by densitometric scanning.
Statistical Analyses
Data are presented as means ± SE. Statistically significant differences between group means were evaluated by ANOVA or the Student's t-test, as indicated in the figure legends. Individual between-group contrasts were evaluated using the Bonferroni test. ![]() |
RESULTS |
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Relative Intestinal CYP27A Level and Drug Inducibility
Figure 1, A and B presents the relative level of CYP27A expression in normal male rat liver and duodenum. As illustrated, the steady-state level of CYP27A mRNA was found to be threefold higher in liver than in duodenum (P < 0.0001). A survey of other parts of the intestine indicates values relative to those found in duodenum of 85, 69, and 77% in jejunum, ileum, and colon, respectively.
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To investigate the modulation of the duodenal CYP27A
transcript by known pharmacological agents, animals were treated with acetone, phenobarbital, -naphtoflavone, 3-methylcholanthrene, and
dexamethasone. As illustrated in Fig. 2,
A and B, only dexamethasone was found to
significantly increase (5-fold induction) CYP27A mRNA
expression in intestine (P < 0.0001). Finally, the
effect of the calcium and D status on the relative abundance of the
CYP27A gene transcript was investigated. As illustrated in
Fig. 2, C and D, CYP27A was found to
be twofold higher in D-depleted than in normal rat duodenum
(P < 0.02), and this was irrespective of the calcium
status of the animals. Indeed, no significant difference was observed
in CYP27A gene expression in duodena obtained from hypocalcemic D-depleted (D
Ca
) compared with that obtained from normocalcemic D-depleted (D
Ca+) rats.
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Effect of D3, 25OHD, or 1,25(OH)2D3 Repletion on CYP27A mRNA Level
The influence of the D status on intestinal CYP27A mRNA abundance prompted investigation on the CYP27A gene response to a 1-wk repletion with either the parent compound D3 or with its hepatic (25OHD) or kidney [1,25(OH)2D3] metabolites.Repletion with D3.
D3 was found to significantly influence CYP27A
mRNA levels, as illustrated in Fig. 3,
with an 80% decrease in the abundance of the CYP27A gene
transcript observed after 1 wk of repletion (P < 0.0001). The dose of D3 used achieved normalization of the circulating Ca2+, 25OHD, and
1,25(OH)2D3 after 1 wk of repletion, as
previously reported (17, 21, 27). The
1,25(OH)2D3 concentrations achieved were found
to be 1,760 ± 152 pmol/l at day 1 of the repletion period and to decrease to 1,115 ± 128 pmol/l at the time of
euthanasia.
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Repletion with 25OHD.
The serum Ca2+ concentrations increased steadily from a
mean value of 0.82 ± 0.01 mmol/l in DCa
to 1.13 ± 0.04 after 1 wk of 25OHD administration. Serum 25OHD concentrations
remained, however, low during the period studied, with a mean
concentration of 6.5 nmol/l after 1 wk of repletion, whereas serum
1,25(OH)2D3 concentrations increased from an
average of 95 ± 15 pmol/l in D-depleted animals to an average of
1,143 ± 110 pmol/l after 1 wk of 25OHD repletion.
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Repletion with 1,25(OH)2D3. The serum Ca2+ concentrations increased steadily from a mean value of 0.73 ± 0.03 to 1.21 ± 0.09 mmol/l throughout the 1 wk of 1,25(OH)2D3 repletion. Serum 25OHD remained unchanged during the course of 1,25(OH)2D3 administration, whereas 1,25(OH)2D3 concentrations which averaged 95 ± 15 pmol/l at day 0 remained at a plateau during the course of 1,25(OH)2D3 repletion, averaging 603 ± 90 pmol/l after 1 day of repletion and 503 ± 123 pmol/l after 1 wk of 1,25(OH)2D3 administration.
As illustrated in Fig. 5, A and B, 1,25(OH)2D3 administration resulted in a highly significant 30% decrease in CYP27A mRNA levels as soon as 24 h after the beginning of 1,25(OH)2D3 administration (P < 0.0008). CYP27A mRNA levels thereafter remained unchanged, with an observed 40% decrease (P < 0.0008) in CYP27A mRNA abundance after 1 wk of 1,25(OH)2D3 exposure compared with the level observed in rat duodena not exposed to the hormone. Moreover, Western analyses (Fig. 5, C and D) revealed a concomitant 31% decrease in CYP27A protein level after 1 wk of 1,25(OH)2D3 administration (P < 0.003).
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25-Hydroxylase activity.
Fig. 6 illustrates the effect of 1-wk
exposure to 28 pmol/day 1,25(OH)2D3 on the
25-hydroxylase activity in isolated duodenal mitochondria.
1,25(OH)2D3 repletion had a significant
influence on CYP27A activity with an average 89% decrease
in 1,25(OH)2D3 production after
incubation with 1
OHD3 (P < 0.01).
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Mechanisms of 1,25(OH)2D3 Action
The observation that the decrease in CYP27A mRNA were highly and rapidly sensitive to 1,25(OH)2D3 prompted studies on the mechanisms by which the hormone influences the abundance of the CYP27A gene transcript. These studies were conducted using an acute model of 1,25(OH)2D3 exposure.Effect of acute 1,25(OH)2D3 administration on the level of the CYP27A gene transcript. Serum Ca2+ concentrations increased only slightly from 0.78 ± 0.01 to a range varying from 0.90 ± 0.01 (2.4 nmol/kg dose) to 1.01 ± 0.03 (240 nmol/kg dose) 6 h after the intravenous injection of 1,25(OH)2D3. The 1,25(OH)2D3 concentrations reached, at the time of euthanasia, were 9,277 ± 551 pmol/l in rats injected with the 2.4 nmol/kg dose to >30,000 pmol/l in animals injected with the 12, 120, and 240 nmol/kg doses of 1,25(OH)2D3 (P < 0.001).
As is illustrated in Fig. 7, A and B, as soon as 6 h after 1,25(OH)2D3 exposure, duodenal CYP27A mRNA levels were found to progressively decrease with increasing doses of the hormone (2.4-240 nmol/kg), whereas CYP24 mRNA levels, which were used as controls for the 1,25(OH)2D3 response, were found to be concomitantly and highly significantly upregulated (Fig. 7, C and D).
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CYP27A mRNA half-life.
As is illustrated in Fig. 8, treatment of
CaD
rats with 0.5 mg/kg actinomycin D did not significantly
influence CYP27A mRNA levels in untreated rats over
the 6-h period studied, although a slight, but not significant,
increase over basal values was observed at the 6-h time period. A dose
of 5 mg/kg actinomycin D, which was also used to verify whether the
dose of actinomycin D used in the original experiment was sufficient to
halt CYP27A gene transcription, revealed that
CYP27A mRNA levels were not influenced for
6 h after
actinomycin D injection compared with untreated Ca
D
controls. All
further studies, therefore, were done using the 0.5 mg/kg dose of
actinomycin D. The effectiveness of the dose of actinomycin D used in
inhibiting the process of transcription was also verified by examining
its effect on the mRNA levels of the gene encoding CYP24. A
0.5 mg/kg dose given 1 h before
1,25(OH)2D3 injection was found to effectively
prevent the upregulation of the CYP24 gene transcript
(results not shown). After the intravenous injection of
1,25(OH)2D3, a 24% decrease in the level of
the CYP27A gene transcript was observed 1 h after actinomycin D administration compared with the level observed in
Ca
D
animals. Moreover, the levels of the transcript steadily decreased to 36% of the level observed in animals not subjected to
actinomycin D administration 6 h after actinomycin D
administration (P < 0.03). Compared with their
actinomycin D-paired D-depleted controls, the relative abundance of the
CYP27A gene transcript was found to be decreased by 77% at
the 6-h time point (P < 0.002).
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CYP27A gene transcription rate.
Nuclear transcription run-on assays were performed on nuclei isolated
from duodena of CaD
rats as well as on nuclei obtained from duodena
of Ca
D
animals exposed to a single 120 nmol/kg intravenous dose of
1,25(OH)2D3 (Fig.
9). The 18S ribosomal gene was used as a
control gene for both the untreated and treated groups. Quantification
for the nuclear run-on assays demonstrated that, within 6 h of
1,25(OH)2D3 exposure, the transcription rate of
the gene encoding CYP27A decreased by 32% compared with the level of expression observed in control duodena. Nonspecific
hybridization, estimated by hybridization to pBS plasmid DNA, did not
account for the observed CYP27A decrease in transcription.
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DISCUSSION |
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Although several studies have reported that the mitochondrial cytochrome P-450 27A is present in multiple sites (14, 31, 34, 37), no study has addressed the regulation of the enzyme in intestine, and the effect of D3 status on the expression of the gene encoding CYP27A in small intestine is also unknown. Indeed, most studies to date have focused on the hepatic regulation of the gene, which has, up to now, been reported to be sensitive to bile acids, glucocorticoids, growth hormone, and insulin (37, 46, 48, 52, 53, 55). Axén et al. (7), on the other hand, have reported that CYP27A located in kidney and liver was affected by 1,25(OH)2D3 administration, stressing that the kidney CYP27A mRNA levels were decreased to a greater extent than those of the liver, an observation which we have confirmed in our laboratory (51). The significance of these observations on the C-25 hydroxylation of D3 have not yet been investigated, although earlier studies have raised the hypothesis that 1,25(OH)2D3 might inhibit the production of 25OHD in human subjects (8). Later studies in the rat indicated, however, that the decrease in serum 25OHD concentrations could be explained mainly by an acceleration in its metabolic clearance rate (15, 27, 28), although species differences in the C-25 hydroxylation of D3 have not been ruled out, the microsomal D3-25-hydroxylase being the predominant enzyme in the rat, whereas, in humans, the mitochondrial CYP27A has been claimed to be the sole D3-25-hydroxylase (44, 45). Our studies show that duodenal CYP27A mRNA levels are significantly lower than those observed in liver but, as in liver (55), the duodenal transcript was shown to be significantly upregulated by dexamethasone, whereas common cytochrome P-450 inducers were shown to be without effect. Data also indicate that, compared with duodena obtained from normal D-replete animals, CYP27A mRNA levels are significantly higher in duodena obtained from D-depleted and that, independently of the circulating Ca2+, clearly indicating that the intestinal CYP27A is highly sensitive to the D status.
Our studies on the effect of D3, 25OHD, and
1,25(OH)2D3 indicate that each compound
significantly lowered the abundance of the CYP27A gene
transcript. A time course of the decrease in CYP27A mRNA
levels after continuous intraperitoneal administration indicates that
the decline in mRNA abundance is gradual, with significant decreases
observed after 72 h of 25OHD repletion and as soon as 24 h
after the initiation of 1,25(OH)2D3 repletion.
CYP27A protein levels and 25-hydroxylase enzyme activity were also
shown to be sensitive to 1,25(OH)2D3.
Furthermore, the studies with the intravenous injection of
1,25(OH)2D3 as well as those on the half-life
of CYP27A mRNA indicate that the gene transcript is rapidly
and dose-dependently downregulated within hours of exposure to the
hormone. These data demonstrate that, in rat duodena, the gene
encoding CYP27A is highly sensitive to the in vivo exposure
to the D3 hormone. Whether 1,25(OH)2D3 is the sole mediator of the
observed downregulation of the CYP27A gene transcript is not
known. Attempts at evaluating the specific role of
1,25(OH)2D3 as opposed to that of 25OHD in the
regulation of CYP27A gene expression were done using
ketoconazole to inhibit the C-1 hydroxylase when 25OHD was
administered. Unfortunately, these attempts were unsuccessful for the
following reasons. 1) Ketoconazole proved to be a
nonspecific monooxygenase inhibitor, inhibiting not only the C-1
(29) but also the C-24 hydroxylase (32, 57),
which resulted in unexpected changes in circulating 1,25(OH)2D3. 2) The effect of
1,25(OH)2D3 on the CYP27A gene
transcript proved to be too sensitive to evaluate subtle changes in the
1,25(OH)2D3 circulating concentrations
induced by ketoconazole. 3)
1,25(OH)2D3 exhibited a rapid effect
[significant inhibition observed within 6 h after iv
1,25(OH)2D3 exposure] to discriminate its role
compared with that of 25OHD, although the time course of inhibition
suggests that the latter is most likely not responsible for the
inhibition observed during the present studies. Although the data
obtained during our studies suggest a highly significant effect of
1,25(OH)2D3 on the downregulation of the
duodenal CYP27A transcript, they do not entirely rule out
the participation of other metabolites such as
24,25(OH)2D3, and/or downstream products of the hormone.
The differences observed in the sensitivity of CYP27A
between animals repleted with D3, 25OHD, or
1,25(OH)2D3 warrants comments. Interestingly,
an earlier study carried out in our laboratory has indicated that the
kinetics of the serum 25OHD and 1,25(OH)2D3 achieved as well as that of the involution of the associated secondary hyperparathyroidism was quite different when animals were repleted with
D3 or with 1,25(OH)2D3
alone (23). Indeed, the serum
1,25(OH)2D3 concentrations achieved with
D3 repletion are much higher (in the 1,500 to 2,000 pmol/l
range, most likely due to the high 1-hydroxylase activity induced by
D depletion) than those achieved when the hormone is applied by
intraperitoneal miniosmotic pump, which, in the present study, proved
to be quite constant, averaging 500 to 600 pmol/l between days 1 and 7 of 1,25(OH)2D3 repletion.
These differences could explain the greater effect of the parent
compound (after 7 days of repletion) on the steady-state expression of the gene encoding CYP27A compared with that observed after
1,25(OH)2D3 administration. However, the in
vivo effect of 1,25(OH)2D3 on CYP27A
mRNA levels is clearly illustrated by the clear dose-response curve
achieved after intravenous administration. A clear effect of
1,25(OH)2D3 on CYP27A mRNA half-life
and on the transcription rate of the gene also supports an action
mediated by the hormone or by immediate and rapidly formed downstream metabolite(s).
The data illustrating that the duodenum (as well as the
jejunum, ileum, and colon) clearly expresses CYP27A as well
as the D3-25-hydroxylase protein lead us to put forward the
hypothesis that a local production of 25OHD can be achieved in the
small intestine. In addition, the already reported presence of
25OHD-1 hydroxylase and 25OHD-24-hydroxylase activities in
intestinal cells as well as in the Caco-2 cell line (5, 16, 36,
47) suggests that D of dietary origin could be locally processed
and transformed into 25OHD, 1,25(OH)2D3, or
24,25(OH)2D/1,24,25(OH)3D. Moreover,
Axén et al. (6) have reported that the
C-27-hydroxylase purified from pig and rabbit livers, as well as
recombinant human CYP27A, was also able to catalyze the
1
-hydroxylation of 25OHD, albeit at a much lower rate than that
observed for the conversion of D3 into 25OHD
(7). Furthermore, CYP27A has also been shown to be active
on other D compounds, such as D2, and 1
OHD3,
as illustrated in the present studies (19, 20, 26).
Collectively, these observations illustrate that a large spectrum of
compounds of the D family can be locally activated by intestinal cells
into active metabolites when taken orally.
The critical elements involved in the
1,25(OH)2D3-mediated downregulation of the
CYP27A gene transcript have not been investigated. Our data
indicate that the mechanisms responsible for the regulation of the gene
involve a decrease in mRNA half-life and a decrease in transcriptional
rate. The effect of calcitriol on the gene, however, is present even in
the absence of normalization of the circulating Ca2+
concentrations, as was illustrated after the intravenous injection of
1,25(OH)2D3, suggesting that Ca2+
is not a critical element in the response to the hormone. In addition,
repletion with calcium alone (which normalizes the serum Ca2+ concentration without affecting the D nutritional or
hormonal status) does not affect CYP27A steady-state mRNA
levels. CYP27A and CYP7 are genes involved in
bile acid biosynthesis. CYP7
is known to be regulated by
some of the orphan receptors mediating the response to fatty acid and
cholesterol such as pregnane X receptor [a nuclear receptor closely
related to vitamin D receptor (VDR) (33)], farsenoid X
receptor, or peroxisome proliferator-activated receptor, which all have
retinoid X receptor (RXR) as a partner for DNA binding. It is
not yet known, however, whether CYP27A is also regulated by
these nuclear receptors and/or what is the role of
1,25(OH)2D3 in these interactions most
particularly in relation with its binding to the VDR and the subsequent
involvement of RXR for DNA binding and activation.
The data obtained during our studies clearly show that the rat duodenum expresses the mitochondrial D3-25-hydroxylase CYP27A. They also show an effect of the D3 nutritional status (D3 and 25OHD) as well as of the D3 hormonal status [1,25(OH)2D3] on the gene mRNA half-life and transcription rate. Thus, in addition to exhibiting high amounts of VDR, the intestine seems to possess the major D3 hydroxylases, indicating that, aside from being able to respond to the classic endocrine actions mediated by 1,25(OH)2D3, the small intestine may exhibit the presence of a fine endocrine/paracrine, or autocrine regulation of D3-related pathways.
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ACKNOWLEDGEMENTS |
---|
The authors are grateful to Manon Livernois for excellent
secretarial assistance. The rabbit polyclonal antibody raised against human CYP27A was provided by Dr. David Russel, University of
Texas Southwestern Medical Center, Dallas, TX. Crystalline reference 25OHD and 1,25(OH)2D3 were gifts from UpJohn,
Kalamazoo, MI, and Hofmann-LaRoche, Nutley, NJ, respectively.
1OHD3 was a gift from Leo Pharma, Ajax, Ontario, Canada.
![]() |
FOOTNOTES |
---|
C. Theodoropoulos was a recipient of a Studentship Award from the McAbbie Foundation. This study was supported by the Medical Research Council of Canada.
Address for reprint requests and other correspondence: M. Gascon-Barré, Centre de recherche, Hôpital Saint-Luc, Centre Hospitalier de l'Université de Montréal, 264 René-Lévesque Blvd. East, Montreal, QC Canada, H2X 1P1 (E-mail: marielle.gascon.barre{at}umontreal.ca).
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.
Received 14 July 2000; accepted in final form 21 March 2001.
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