1,25-Dihydroxyvitamin D3 downregulates the rat intestinal vitamin D3-25-hydroxylase CYP27A

Catherine Theodoropoulos, Christian Demers, Ali Mirshahi, and Marielle Gascon-Barré

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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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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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-1alpha 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 1alpha -hydroxyvitamin D3 (1alpha 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-1alpha (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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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 (D-Ca-) 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), beta -naphtoflavone (3 daily ip injections: 80 mg/kg), acetone (1% vol/vol in drinking water for a period of 10 days), or phenobarbital sodium (350 mg/ml in drinking water for a period of 10 days) (40).

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 D-Ca- rats or from rats exposed to a single intravenous dose of 120 nmol/kg 1,25(OH)2D3 6 h before euthanasia. Nuclear run-on assays were done as indicated below.

25-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. 1alpha -hydroxyvitamin D3 (1alpha OHD3) (Leo Pharma, Ajax, ON, Canada) was used as substrate.

Duodenal 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 1alpha OHD3 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 1alpha 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.

Total intestinal RNA was extracted, blotted onto nylon membranes (Qiagen, Mississauga, ON, Canada) and processed for Northern analyses as previously described (35). The radiolabeled CYP27A probe was a cDNA fragment generated specifically for the present studies by RT-PCR. The 404 bases corresponded to base pairs 399-803 within the NH2-terminal coding region of the rat gene on the basis of the published sequence of Su et al. (49), (GenBank accession no. M38566). The primers 5'-TCTCTGGCTCTAAACTCTTGGC-3' and 5'-CTCGTGAAGTGCAGCACATA-3' used were custom synthesized by the Sheldon Biotechonology Centre (McGill University, Montreal, QC, Canada). The following PCR program was used for 30 cycles: 30 s at 65°C, 10 s at 72°C, and 30 s at 95°C. The fragment obtained was cloned into the vector PCRII (Invitrogen, Carlsbad, CA) and sequenced to confirm identity. The functional specificity of the CYP27A fragment generated revealed higher mRNA expression in female than in male liver and a significant hepatic induction of the gene transcript by dexamethasone (50) as expected from previous studies (1, 2, 4, 48).

The CYP24 probe was a 247-base rat renal cDNA insert from the KpnI site of the pUC19 vector (38), and the 18S ribosomal RNA was a 1.5-kb human cDNA insert from the EcoRI site of pBluescript (pBS) SK- vector (American Tissue Culture Collection no. 77242). Probe labeling, blot hybridization, washing, exposure, and photodensitometric evaluation were performed as previously described (18, 35).

Nuclear run-on transcriptional assay. Nuclei were isolated from duodena of D-Ca- 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.


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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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Fig. 1.   A: Northern analysis representing the relative abundance of the CYP27A gene transcript in male liver and duodenum. B: means ± SE of CYP27A mRNA levels observed in 4 animals/group. Statistically significant differences between group means were analyzed by Student's t-test.

To investigate the modulation of the duodenal CYP27A transcript by known pharmacological agents, animals were treated with acetone, phenobarbital, beta -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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Fig. 2.   Influence of cytochrome P-450 inducers on the duodenal expression of the gene encoding CYP27A. A and C: representative Northern analyses of the CYP27A gene transcript. B: evaluation of CYP27A mRNA levels in normal control male rat duodena and after administration of cytochrome P-450 inducers; n = 3 animals/group. D: evaluation of the relative level of the CYP27A gene transcript in normal and in hypocalcemic (Ca-; serum Ca2+: 0.78 ± 0.02) and normocalcemic (Ca+; serum Ca2+: 1.26 ± 0.02) vitamin D-depleted (D-) male rat duodena. Data are presented as means ± SE; n = 11 for control, n = 15 for D-Ca-, and n = 3 for D-Ca+. Statistically significant differences between group means were analyzed by ANOVA (cytochrome P-450 induction studies) and by the Student's t-test (normal and D-Ca- or D-Ca+ studies).

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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Fig. 3.   Influence of D3 repletion on the duodenal expression of the gene encoding CYP27A. A: representative Northern analysis of the CYP27A gene transcript in D-Ca-, and after 7 days of D3 repletion by miniosmotic pump (ip) at a dose of 6.5 nmol/day. B: steady-state levels of CYP27A mRNA in D-Ca- and in D3-repleted rat duodena. Data are presented as means ± SE; n = 5 animals/group. Statistically significant differences between group means were analyzed by Student's t-test.

Repletion with 25OHD. The serum Ca2+ concentrations increased steadily from a mean value of 0.82 ± 0.01 mmol/l in D-Ca- 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.

As illustrated in Fig. 4, the intestinal CYP27A gene transcript exhibited a progressive decrease in its level of expression with a nonsignificant 10% decrease at day 1 but with a significant 33% decrease after 72 h of 25OHD administration (P < 0.03). After 1 wk of 25OHD repletion, the mean level of the CYP27A transcript was found to be decreased by 55% compared with the level observed in animals not exposed to 25OHD (P < 0.008). Moreover, the decrease in CYP27A mRNA abundance was shown to be linear over the 1-wk period studied (r2 = 0.787, P < 0.001).


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Fig. 4.   Influence of 25-hydroxyvitamin D (25OHD) repletion on the duodenal expression of the gene encoding CYP27A. A: representative Northern analysis of the CYP27A gene transcript in D-Ca-, and after 1, 3, 5, or 7 days of 25OHD repletion by miniosmotic pump (ip) at a dose of 28 pmol/day. B: steady-state levels of CYP27A mRNA in D-Ca- and in 25OHD3-repleted rat duodena. Data are presented as means ± SE; n = 3 animals/group. Statistically significant differences between group means were analyzed by ANOVA, with individual contrasts evaluated by the Bonferonni test. Main effect, P < 0.001; significantly different from D-Ca-, *P < 0.003; **P < 0.002, ***P < 0.0008.

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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Fig. 5.   Influence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] repletion on the duodenal expression of the gene encoding CYP27A and on the level of the CYP27A protein. A: representative Northern analysis of the CYP27A gene transcript in D-Ca- and after 1, 3, 5, or 7 days of 1,25(OH)2D3 repletion by miniosmotic pump (ip) at a dose of 28 pmol/day. B: steady-state level of CYP27A mRNA in D-Ca- and in 1,25(OH)2D3-repleted rat duodena. C: representative Western analysis of the CYP27A protein in D-Ca- and 7 days after 1,25(OH)2D3 repletion. D: steady-state level of CYP27A protein in D-Ca- and in 1,25(OH)2D3-repleted rat duodena. Data are presented as means ± SE; n = 3 animals/group. Statistically significant differences between group means were analyzed by ANOVA, with individual contrasts evaluated by the Bonferonni test. Main effect, P < 0.0002; significantly different from D-Ca-, ***P < 0.0008.

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 1alpha ,25(OH)2D3 production after incubation with 1alpha OHD3 (P < 0.01).


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Fig. 6.   Mitochondrial 25-hydroxylase activity from freshly isolated duodenal mucosal cells obtained from D-Ca- (n = 2) or 1,25(OH)2D3-repleted rats (n = 3). Statistically significant differences between group means were analyzed by Student's t-test.

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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Fig. 7.   Influence of acute 1,25(OH)2D3 administration on the expression of the genes encoding CYP27A and CYP24. All animals received a single iv dose of 0, 2.4, 12, 120, or 240 nmol/kg and were killed 6 h later. A: representative Northern analysis of the CYP27A gene transcript in D-Ca- and in animals exposed to 1,25(OH)2D3. B: steady-state levels of CYP27A mRNA in duodena of D-Ca- and of animals injected with 1,25(OH)2D3. C: representative Northern analysis of the CYP24 gene transcript in D-Ca- and in animals exposed to 1,25(OH)2D3. D: steady-state levels of CYP24 mRNA in duodena of D-Ca- and of animals injected with 1,25(OH)2D3; n = 3 animals/group. Statistically significant differences between group means were analyzed by ANOVA, with individual contrasts evaluated by the Bonferroni test. CYP27A mRNA levels: main effect, P < 0.003. Statistically different from D-Ca-, *P < 0.03; CYP24 mRNA levels: main effect, P < 0.0001. Statistically different from D-Ca-, ***P < 0.0008.

CYP27A mRNA half-life. As is illustrated in Fig. 8, treatment of Ca-D- 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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Fig. 8.   In vivo half-life of CYP27A mRNA in duodena of D-Ca- and 1,25(OH)2D3-injected rats. Animals received a single iv dose (120 nmol/kg) of either 1,25(OH)2D3 or vehicle 3 h before actinomycin D (0.5 mg/kg) administration . Animals were killed 1, 3, or 6 h after actinomycin D administration.  CYP27A mRNA levels in D-Ca- rat duodena,  CYP27A mRNA levels in 1,25(OH)2D3-injected rat duodena. Data are presented as means ± SE; n = 2 animals/group. Statistically significant differences between group means were analyzed by ANOVA, with individual contrasts evaluated by the Bonferonni test. Main effect, P < 0.04; Significantly different from D-Ca-, *P < 0.03.

CYP27A gene transcription rate. Nuclear transcription run-on assays were performed on nuclei isolated from duodena of Ca-D- 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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Fig. 9.   Rate of transcription of the CYP27A gene transcript in duodena of D-Ca- and 1,25(OH)2D3-injected rats. Animals received a single iv dose (120 nmol/kg) of 1,25(OH)2D3 6 h before euthanasia. A: representative nuclear run-on transcriptional assays. B: quantitative evaluation of the transcriptional run-on assays was achieved by scanning densitometry. Transcriptional activity was measured in 3 different experiments with 2-3 rats/experiment for each group. Data are presented as means ± SE. Statistically significant differences between group means were analyzed by Student's t-test.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-1alpha 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-1alpha (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 1alpha -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-1alpha 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 1alpha -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 1alpha 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 CYP7alpha are genes involved in bile acid biosynthesis. CYP7alpha 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.


    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. 1alpha OHD3 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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Am J Physiol Endocrinol Metab 281(2):E315-E325
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