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Original Studies |
-Hydroxylase Gene Transcription Product in Cultures of Human Syncytiotrophoblast Cells1
Department of Reproductive Biology, Instituto Nacional de la Nutrición Salvador Zubirán, México City 14000, México
Address correspondence and requests for reprints to: Fernando Larrea, M.D., Department of Reproductive Biology, Instituto Nacional de la Nutrición Salvador Zubirán, Vasco de Quiroga No. 15, México 14000, México D.F. E-mail: larrea{at}mailer.main.conacyt.mx
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase (1-(OH)ase) gene expression products
in cultured human syncytiotrophoblast. Total RNA was isolated from
cultured placental cells and subjected to Northern blots or RT-PCR by
using 1-(OH)ase-specific primers. The amplified complementary DNA
fragments were analyzed by gel electrophoresis and nucleotide
sequencing. Total RNA from kidney HEK 293 cells was subjected to
reverse transcriptase reaction, and a 298-bp complementary DNA
1-(OH)ase probe was generated by PCR. Primary cultures of human
syncytiotrophoblasts exhibited 1-(OH)ase activity, and a transcript
for this gene could be demonstrated in these cells. Northern blot
analysis revealed the presence of a 2.5-kb product, similar in size to
that previously reported in kidney. RT-PCR analysis demonstrated the
presence of a single transcript with nucleotide sequence identical to
that previously reported for human 1-(OH)ase complementary DNA
clones. In addition, data are presented which suggest that
differentiation of cytotrophoblast to the syncytial state was not
necessary for this gene to be expressed, which may indicate a role of
this enzyme all through pregnancy. The overall results of this study
provide evidence for the presence of 1-(OH)ase in the human
placenta, suggesting that conversion of 25OHD3 to
1,25-dihydroxyvitamin D3 in the trophoblast is most
probably attributed to an enzymatic 1-hydroxylation reaction. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase
(1-(OH)ase), a mitochondrial cytochrome P450
enzyme, with a key role in calcium homeostasis. The first observation
leading to the establishment of an extrarenal source of 1-(OH)ase
was in pregnant rats, where bilateral nephrectomy reduced, but did not
completely eliminate, the serum concentrations of
1,25-(OH)2D3 (3). In fact,
it has been shown that decidual cells represent a site of calcitriol
synthesis during pregnancy (4). Although in vitro studies
provided evidence that, in addition to human decidua, human and rodent
placental trophoblasts produced
1,25-(OH)2D3 (3, 4), a
number of investigators have been unable to demonstrate a consistent
and detectable production of calcitriol by these cells (5, 6).
Similarly, Hollis et al. (7) have suggested that
1,25-(OH)2D3 produced by
human placenta, under in vitro conditions, is the result of
a free radical chemistry, rather than an enzymatic-driven
1-hydroxylation reaction. In contrast, and in agreement with
earlier observations (8, 9), we have recently shown (10) that cultured
human syncytiotrophoblast cells were able to produce
1,25-(OH)2D3 when incubated
in the presence of physiological concentrations of 25-hydroxyvitamin
D3 (25OHD3). This
conversion was significantly stimulated, in a dose-dependent
manner, by the presence of the insulin-like growth factor I (IGF-I) and
inhibited with the protein synthesis inhibitor cycloheximide,
suggesting the existence of a local protein-dependent regulatory
effect. Taken together, these data suggest that human placenta is able
to synthesize 1,25-(OH)2D3
from its endogenous precursor by an enzymatic 1- hydroxylation
mechanism. These findings are of importance, because 1-(OH)ase gene
expression has not yet been detectable in the human placenta (11, 12, 13).
Nowadays, there is little, if any, information on the molecular
mechanisms underlying placental
1,25-(OH)2D3 production and
its hormonal regulation, including the understanding of its biological
significance. Herein, we report the presence, in cultured human
syncytiotrophoblast cells, of a 1-(OH)ase gene transcription product
with nucleotide sequence identical to that of transcripts previously
characterized in the human kidney. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DMEM and DMEM-F12, HBSS, FCS, HEPES, streptomycin, and Fungizone were obtained from Life Technologies, Inc. (Grand Island, NY). Percoll, 8-bromo adenosine 3',5'-cyclic monophosphate (8-Br-cAMP), deoxyribonuclease I, BSA, glutamine, cycloheximide, and trypsin were purchased from Sigma (St. Louis, MO). All solvents [high-pressure liquid chromatography (HPLC) grade] were obtained from Merck & Co., Inc. (Darmstadt, Germany). Unlabeled authentic 25OHD3 and 1,25-(OH)2D3 were a generous gift from Dr. E. M. Gutknecht and Dr. P. Weber (F. Hoffmann-La Roche LTD, Basel, Switzerland). The 25-hydroxy-[26,(27)-methyl-3H]cholecalciferol ([3H]25-(OH)D3; SA, 17 Ci/mmol) was purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). NIDDK (Rockville, MD) kindly provided human CG (hCG) RIA. All other reagents were of analytical grade.
Tissue preparation and cell culture
The study protocol was approved by the Human Ethical Committee of the Institute. Term placentae (38–42 weeks of gestation) were obtained from normal pregnant women after spontaneous vaginal delivery. Tissues were brought immediately to the laboratory, where several cotyledons were removed and rinsed thoroughly in 0.9% NaCl at room temperature. The isolation and culture of cytotrophoblasts was performed as described by Kliman et al. (14). Briefly, soft villous tissue (30 g), free of connective tissue and vessels, was collected. Tissue was coarsely minced and digested with 0.125% trypsin and 0.2 mg/mL deoxyribonuclease I (1, 500 Kunitz units/mg) in warmed calcium- and magnesium-free HBSS, containing 25 mmol/L HEPES (pH 7.4), for 30 min at 37 C. Cell suspensions were pooled, centrifuged at 1000 x g for 10 min, and resuspended in DMEM containing 25 mmol/L HEPES and 25 mmol/L glucose (DMEM-HG). The resultant cell suspension was placed on 5–70% Percoll (vol/vol) gradients made up in HBSS. Gradients, which consisted of 5% steps of 3 mL each, were centrifuged at 1200 x g at room temperature for 20 min. After centrifugation, the middle band (density, 1.048–1.062), containing the cytotrophoblasts, was removed, washed once with DMEM-HG, and resuspended in medium for tissue culture. Percoll gradient purified cytotrophoblasts were diluted to a concentration of 2 x 106 cells/mL with DMEM-HG containing 4 mmol/L glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.25 µg Fungizone/mL, and 20% heat-inactivated FCS, plated in 35-mm Nunclon culture dishes (Nunc, Roskilde, Denmark), and incubated in humidified 5% CO2-95% air at 37 C. After 2 days in culture, or otherwise indicated, cells were incubated in serum-free DMEM-F12 with low calcium and phosphate concentrations.
Daily, the morphological aspects of cell cultures were examined. Human
chorionic gonadotropin in the culture media was measured, as previously
described (15), by specific RIA using reagents and protocols provided
by the NIDDK. Anti-hCG-H80, at a final working dilution of 1:150 000,
was used as antiserum. This antiserum exhibits 1.2 and 3.2%
cross-reactivities with free hCG - and ß-subunits, respectively.
The sensitivity of the assay was 0.025 ng/tube, and the inter- and
intraassay coefficients of variation were <10 and <6%, respectively.
Total protein content of cell cultures was measured by the method of
Lowry et al. (16) using BSA as standard.
Complementary DNA (cDNA) synthesis and PCR amplifications
Total RNA was isolated from cultured syncytiotrophoblast cells,
as described by Chomczynski and Sacchi (17). One µg of
total RNA was used as template for cDNA synthesis using the
SuperScript preamplification system (Life Technologies).
PCR amplification was then performed using Taq polymerase
and the following primers:
5'-ACGCTGTTGACCATGGC-3' for the sense primer, and
5'-GTGACACAGAGTGACCAGCGTAT-3' for the
antisense primer. These primers generated a 543-bp 1-(OH)ase RT-PCR
product. The PCR products were resolved on agarose gels, eluted,
subjected to further amplification, and purified through Centricon-30
membranes (Amicon, Beverly, MA) for sequence analysis. To monitor
efficiency for RT reaction, we used, as a control, the amplification of
the ubiquitous protein cyclophilin with the following sense and
antisense primers: 5'-CCCCACCGTGTTCTTCGACAT-3',
and 5'-AGGTCCTTACCGTTCTGGTCG-3' which yielded a
453-bp RT-PCR product. All oligonucleotides were synthesized in a DNA
synthesizer Model 391 (PE Applied Biosystems,
Perkin-Elmer Corp. Cetus Co., Norwalk, CT). PCR
amplifications were performed on a Perkin-Elmer Corp.
Cetus 9600 DNA Thermal Cycler using the following program: a
denaturation step at 94 C for 1 min, followed by 30 cycles at 94 C for
50 sec, 60 C for 50 sec, and 72 C for 1 min. Finally, a 7-min extension
period at 72 C was performed. Incubations, in the absence of reverse
transcriptase, were used as controls for RT-PCR. The bands of predicted
size (543-bp) were also confirmed as human 1-(OH)ase, by Southern
blot analysis of previously separated DNA on 1.2% agarose gels with a
human 1-(OH)ase cDNA probe (298-bp) radiolabeled with
[32P]deoxycycidine triphosphate
([32P]dCTP) by random priming. This probe was
obtained from human embryonic kidney cells (HEK 293, ATCC CRL-1573,
Microbix Biosystems, Ontario, Canada) by RT-PCR, as described above,
using the following sense and antisense primers:
5'-GTTGCTATTGGCGGGAGTGGAC-3' and
5'-GTGACACAGAGTGACCAGCGTAT-3', respectively.
Sequence analysis
Both DNA RT-PCR strands from human placenta and kidney cells were sequenced by ABI PRISM Dye Terminator Cycle Sequencing Kit (Perkin-Elmer Corp., Foster City, CA) with 25 cycles at 96 C for 10 sec, 50 C for 5 sec, and 60 C for 4 min on a Perkin-Elmer Corp. Cetus 9600 DNA Thermal Cycler. Then samples were dried in a speed-vac, resuspended in sequence loading buffer, denatured at 95 C for 5 min, and loaded on a 4.75% polyacrylamide gel. The sequence determination was carried out using the DNA sequencer model 373–01 (PE Applied Biosystems-Perkin-Elmer Corp.).
Characterization of the 1-(OH)ase messenger RNA (mRNA)
For Northern blots, 30 µg of total cellular RNA was size-fractionated on a 1.2% formaldehyde-agarose gel. After electrophoresis, RNA was transferred into Zeta probe membranes (Bio-Rad Laboratories, Inc. New York, NY) by capillary diffusion, fixed by UV cross-linking, and probed with the 298-bp [32P]dCTP-labeled cDNA fragment obtained from human HEK 293 cells. After prehybridization for 1 h at 68 C, the radioactive probe was added and hybridized in 0.25 mol/L Na2HPO4 and 7% SDS, during 18 h at 68 C.
Activity of 1-(OH)ase in human placental cell cultures
To assess the ability of syncytiotrophoblast cells to convert 25OHD3 into 1,25-(OH)2D3, we carried out experiments in incubations of placental cells on the third day of culture. At this time, medium was changed, and cells were incubated in 2 mL serum-free medium (DMEM-F12) in the presence of [3H]25OHD3, at a final concentration of 5 nmol/L, during 120 min. Culture medium was then transferred to glass tubes, and cells were washed with 1 mL methanol. Protein cell content was determined after addition of 0.5 mL of 1 mol/L NaOH. The [3H]25OHD3 and its metabolites were extracted from the medium with an additional 3 mL methanol, followed by 4 mL chloroform (10, 18). The chloroform phase was dried down under N2, and lipidic extracts were redissolved in chromatographic solvent. The samples were cochromatographed with 0.1 µg unlabeled authentic 1,25-(OH)2D3 as elution marker on a Waters HPLC fitted with a photodiode array detector (PDA; model 996; Waters Corp. Associates, Milford, MA), using an ultrasphere Si, 5 µm, 4.6 x 250 mm column (Beckman Coulter, Inc., Palo Alto, CA). Two-step straight-phase HPLCs were used to separate the vitamin D3 metabolites (10, 18). The conversion rate of [3H]25OHD3 into putative [3H]1,25-(OH)2D3 was determined by calculating the percentage of radioactivity coeluting with authentic unlabeled 1,25-(OH)2D3 after the two successive HPLCs. Results were expressed as fmol/mg protein.
Statistical analysis
Data are presented as the mean ± SD. All
experiments were performed at least three times. Statistical
significance was established using Student’s t test.
P 
0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Microscopic examination of cell cultures showed that, within 3
days after plating, the cultured cytotrophoblasts formed cell
aggregates conformed mostly (99%) of larger areas containing multiple
nuclei. In addition, single mononuclear cells were not observed at this
time of culture. By day 3, multinucleated cells seemed to form a
network (Fig. 1
) that corresponded to
functional syncytiotrophoblasts, in terms of their ability to secrete
placental hCG. Figure 1 shows the data of three experiments on the
temporal pattern of hCG release from cultured trophoblast cells. In
each case, little or no detectable hormone was present in the culture
medium during the first day of plating, regardless of the presence of
8-Br-cAMP. A detectable and significant (P < 0.01)
increase in the level of hCG was observed by day 2 in
8-Br-cAMP-stimulated cultures, reaching peak values between days 3 and
4 and then decreasing daily throughout the remaining days. As can be
seen, addition of 8-Br-cAMP significantly increased (P
< 0.001) hCG secretion, when compared with cultures in the absence of
the cyclic nucleotide analogue.
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-(OH)ase mRNA by syncytiotrophoblast
cells
Because initial attempts to identify placental 1-(OH)ase gene
products from total RNA isolated from fresh placental tissue were
unsuccessful, we decided to use Percoll gradient-purified
cytotrophoblast cells kept in culture as a source of placental RNA.
Gene expression was evaluated by Northern blot analysis using a 298-bp
cDNA fragment for 1-(OH)ase gene obtained from kidney HEK 293 cells,
as described under Materials and Methods and shown in Fig. 2A. Figure 2B shows a Northern blot
analysis of total cellular RNA isolated on day 3 of culture. From the
Northern blot analysis, a signal that corresponds to 1-(OH)ase mRNA
was found in the syncytiotrophoblast cells (lanes 2–4). A
similar-sized (2.5-kb) 1-(OH)ase transcript was found in kidney HEK
293 cells (lane 1). Decidua total RNA was probed and used as positive
control (lane 5). RT-PCR and DNA sequencing further confirmed these
results.
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RT-PCR was performed using primers based on human
P450 1-(OH)ase cDNA sequence (11). RT-PCR of
RNA from syncytiotrophoblast and HEK 293 cells yielded, on Southern
blots, a single cDNA band of the expected size (543-bp) for the
oligonucleotide primers used (Fig. 3, lanes 2 and 4, respectively). In the absence of RT, none of the RNA
samples from syncytiotrophoblast cells subjected to PCR for
1-(OH)ase gave positive results (Fig. 3, lane 3). Similar results
were obtained when human genomic DNA was used instead of RNA (Fig. 3, lane 1).
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, the nucleotide
sequence of the 543-bp fragment from syncytiotrophoblast cells was
found to be 100% identical, over all the nucleotides flanked by the
PCR primers, to the nucleotide sequences of human 25-hydroxyvitamin
D3 1-hydroxylase cDNA clones contained in
GeneBank [accession numbers AB005038 (11), AF020192 (12), and AB005989
(13)]. The complete 1-(OH)ase cDNA sequence consists of 2469 bp, so
the fragment reported herein, in the placenta, is located within 706-
and 1248-bp sections of the full-length cDNA. Identical results
were obtained when the 543-bp RT-PCR fragment from HEK 293 cells was
sequenced (data not shown).
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-(OH)ase mRNA and enzyme activity in
syncytiotrophoblasts cultures
To ascertain the pattern of expression of 1-(OH)ase mRNA
throughout culture, total cellular RNA was obtained at different times
from plating (24, 48, 72, and 96 h) and prepared for RT and PCR
amplifications using specific primers. Total RNA was also extracted
from mononuclear Percoll gradient-purified cytotrophoblast cells and
subjected to RT-PCR with the same set of primers (time zero of
culture). As shown in Fig. 5, it was
demonstrated that 1-(OH)ase mRNA is expressed at all culture times
studied, including those in the less-differentiated Percoll
gradient-purified cytotrophoblast cells taken as representatives of day
zero of culture. In addition, relative abundance of 1-(OH)ase mRNA
was obtained by normalizing the 543-bp band intensity (Fig. 5B) with
that generated for the constitutive gene cyclophilin (Fig. 5C). Despite
the absence of an apparent temporal pattern of expression of
1-(OH)ase mRNA throughout culture (Fig. 5A), Northern blots of total
cellular RNA, obtained at different days of plating, showed a gradual
increase of expression, up to 96 h of culture (Fig. 6A). This difference may be explained by
the fact that nonquantitative RT-PCR was used for temporal mRNA
expression in cultured placenta.
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-(OH)ase was assessed in placental cultures by their
ability to convert
[3H]25OHD3 into
[3H]1,25-(OH)2D3.
Cells at various times of plating were incubated in the presence of
substrate. After 2 h of incubation, analysis of samples, using
two-step straight-phase HPLC, showed the presence of a more polar
metabolite that coeluted as a single peak with authentic unlabeled
1,25-(OH)2D3 (data not
shown). As depicted in Fig. 6B
, the amount of putative
[3H]1,25-(OH)2D3
formed by syncytiotrophoblast cells significantly increased
(P < 0.001) at the second and third day of
culture. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To prove that the P450 1-(OH)ase gene is expressed in the human
placenta, we sought to investigate the presence of P450 1-(OH)ase
mRNA in cultures of human syncytiotrophoblasts obtained from normal
term placentas. This culture system has extensively been proven to form
functional syncytiotrophoblasts free of mononuclear fibroblast cells
(14, 16). RT-PCR was used in this study because the activity and,
probably, the content of mRNA of placental 1-(OH)ase, as in the case
of kidney, are very low. In fact, in recent communications from two
laboratories (11, 12), expression of this gene could not be detected by
Northern blots using total RNA isolated from fresh human placental
tissue. Herein, we report the presence of a transcriptional product of
1-(OH)ase in cultures of human syncytiotrophoblast cells with a
nucleotide sequence identical to human kidney 1-(OH)ase (11).
Expression of human and rat P450 1-(OH)ase
cDNA in mammalian cells has been reported (12, 26). Transfection of a
plasmid expressing the full-length cDNA into cultured mouse Leydig
MA-10 and monkey kidney COS-7 cells resulted in a marked 1-(OH)ase
activity, thus providing evidence that cloned cDNA encoded the P450
1-(OH)ase, with robust enzymatic activity. The gene for human
1-(OH)ase spans approximately 6 kb, is composed of nine exons, and
is present in a single copy (11). In addition, it shares a relatively
high homology with vitamin D3 25-(OH)ase, and the
deduced amino acid sequence shows 82% homology with the rat enzyme
(26). Interestingly, although there have been few reports on the
purification and antibody preparation against 1-(OH)ase (27), there
is not yet a subsequent definitive structural protein characterization,
and availability of specific antibodies against human kidney
1-hydroxylase is still lacking.
In this study, analysis of temporal expression of 1-(OH)ase mRNA in
cultured syncytiotrophoblast cells revealed the presence of a single
expected-size RT-PCR product in either Percoll gradient-purified
trophoblasts or in cultured differentiated syncytiotrophoblast cells.
This finding indicates that, in this in vitro system,
differentiation to a syncytial state was not necessary for this gene to
be expressed, and this suggests a possible role of this enzyme all
through pregnancy. Similar observations have been previously reported
for other well-characterized trophoblast products under the same
culture conditions (14). In addition, the expression of mRNA for human
1-(OH)ase, determined by Northern blot analysis from different
sources, demonstrated a major transcript of approximately 2.5 kb in
kidney and decidua. Furthermore, Northern blot analysis of cultured
syncytiotrophoblasts mRNA revealed a single transcript of similar size
as, but in a considerably lower amount than, the one found in both
human kidney and decidua. These findings agree with previous reports on
the size, tissue distribution, and abundance of 1-(OH)ase mRNA (11, 12), allowing us to establish, for the first time, the presence of an
mRNA transcript of this enzyme in the human trophoblast, similar in
size to that previously described in the human kidney. Furthermore,
these observations strongly support the concept that, as in the case of
kidney and decidua, the placenta enzyme is also encoded by the same
gene.
Although transcriptional regulation of the 1-(OH)ase gene has not
been investigated in placental cells, it is possible, as reported
recently (10), that expression of this gene in the placenta could be
regulated similarly to the one present in the kidney. Thus, vitamin D
status and those factors known to influence the enzyme expression and
activity in the kidney should be considered among potential candidates
in regulating also placental 1-(OH)ase. In addition, expression of
1-(OH)ase mRNA in trophoblast and decidual cells, together with the
recent purification and characterization of
1,25-(OH)2D3 receptor from
human placenta (28), may suggest a local-tissue-specific function of
calcitriol during pregnancy. Thus, the biological function of
1,25-(OH)2D3 in the
fetoplacental unit may be considered either endocrine or
autocrine/paracrine in nature, depending on its site of synthesis.
Furthermore, that placenta contributes to
1,25-(OH)2D3 serum
concentrations during pregnancy is derived from a number of case
reports in patients with pseudohypoparathyroidism (PsHP) who remained
normocalcemic without calcitriol treatment during pregnancy (29). These
observations, taken together with those demonstrating that placental
synthesis of 1,25-(OH)2D3
is not affected in patients with PsHP (30), may indicate that
calcitriol treatment in both PsHP and hypoparathyroid patients during
pregnancy should be adapted to physiological needs, to keep calcium
levels in the normal range, as previously reported (31, 32, 33).
Inasmuch as the results presented herein should be only interpreted as
indicating that human placenta expresses 1-(OH)ase mRNA, the
in vitro-observed 1-hydroxylase activity in this and
other studies may, in part, contribute to establish the local
production of the protein. In addition, the overall data support and
extend recent observations from our laboratory, suggesting that
conversion of [3H]25OHD3
to
[3H]1,25-(OH)2D3
in cultured placenta is attributed to an enzymatic 1-hydroxylation
reaction.
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Acknowledgments |
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Footnotes |
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Received February 9, 2000.
Revised March 29, 2000.
Accepted April 6, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-Dihydroxyvitamin D3
and 24,25-dihydroxyvitamin D3 in vitro
synthesis by human decidua and placenta. Nature. 281:317–319.[CrossRef][Medline]
,25-dihydroxyviytamin D3:biochemical characterization
and production in normal subjects and patients with
pseudohypoparathyroidism. J Clin Endocrinol Metab. 62:192–196.[Abstract]
-hydroxylase. Biochem Biophys Res Commun. 239:527–533.[CrossRef][Medline]
-hydroxylase and mutations causing
vitamin D-dependent rickets type 1. Mol Endocrinol. 11:1961–1970.-hydroxylase and vitamin D synthesis. Science. 277:1827–1830.-hydroxylase cDNA. Proc Natl Acad Sci USA. 94:12920–12925.,25-dihydroxyvitamin D3: biochemical characterization
and production in normal subjects and patients with
pseudohypoparathyroidism. J Clin Endocrinol Metab. 62:192–196.
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) or absence (
) of 8-Br-cAMP was removed and assayed
for hCG, as described in Materials and Methods.
Bars represent the mean ± SD of three
separated cultures. The top panel shows the in
vitro differentiation of cytotrophoblasts. Cells were fixed at
the first, second, and third day of culture, respectively, and stained
with hematoxylin. a, P < 0.001 vs.
without 8-bromo-cAMP; b, P < 0.01
vs. day 1.
