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Expression and Activity of 25-Hydroxyvitamin D-1-Hydroxylase Are Restricted in Cultures of Human Syncytiotrophoblast Cells from Preeclamptic Pregnancies

Department of Reproductive Biology, National Institute of Medical Sciences and Nutrition Salvador Zubirán, México Distrito Federal, 14000, México

Address all correspondence and requests for reprints to: Fernando Larrea, M.D., Department of Reproductive Biology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga No. 15, México Distrito Federal, 14000, México. E-mail: . larrea{at}sniconacyt.mx

Abstract

The human placenta synthesizes 1,25-dihydroxyvitamin D₃ and expresses the vitamin D receptor. Because preeclampsia (PE) is associated with low circulating levels of maternal 1,25-dihydroxyvitamin D₃ and IGF-I, it is possible that alterations in calcium metabolism seen in PE could occur at the level of the fetoplacental unit. In this study, the patterns of gene expression and enzyme activity of 25-hydroxyvitamin D-1-hydroxylase (1-hydroxylase) and the abundance of IGF-I mRNA in placentas from normal (NT) and PE-complicated pregnancies were investigated. Cultured syncytiotrophoblast cells from preeclamptic placentas had only one tenth the activity of 1-hydroxylase and did not respond to IGF-I, when compared with NT cultures. Similarly, the levels of 1-hydroxylase mRNA in syncytiotrophoblast cells from PE placentas under basal and IGF-I-stimulated conditions were significantly reduced. In contrast, IGF-I mRNA levels were found to increase during the differentiation process, with no differences between NT and PE cultures. These results support the role of placenta as a contributor to the abnormalities observed in calcium metabolism in PE.

ABNORMALITIES IN CALCIUM metabolism have been involved in the pathophysiology of pregnancy-induced hypertension (1, 2), which have also been linked to preeclampsia (PE)/eclampsia (3, 4, 5). PE is a common disease and remains as a major cause of maternal morbidity and mortality in developing countries. We previously reported that PE is associated with low circulating levels of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] and IGF-I in both maternal and umbilical cord compartments (6). Furthermore, results from this laboratory have established that placenta expresses the mitochondrial cytochrome P₄₅₀ 25-hydroxyvitamin D-1-hydroxylase (1-hydroxylase) gene, which agreed with the capability of the placenta to produce 1,25(OH)₂D₃ (7). In addition, placental 1-hydroxylase activity, as in the case of kidney (8, 9), was stimulated in a dose-dependent manner by IGF-I (10), suggesting that this growth factor acts as a physiological regulator of placental 1,25(OH)₂D₃ production. Therefore, we hypothesized that alterations in calcium metabolism that occur in PE as a result of low 1,25(OH)₂D₃ are attributable, in part, to an altered 1-hydroxylase expression and/or regulation at the level of the fetoplacental unit.

Herein, we report a study aimed at investigating the hormonal basis for low 1,25(OH)₂D₃ circulating levels in PE. We determined the activity and expression of the 1-hydroxylase enzyme in cultured placental syncytiotrophoblast cells obtained from normal (NT) and PE complicated pregnancies. In addition, expression of IGF-I gene, during trophoblast differentiation in both NT and PE cell cultures, was also studied.

Subjects and Methods

Subjects

The study protocol was approved by the Human Ethical Committee of the National Institute of Medical Sciences and Nutrition Salvador Zubirán. Subjects were considered to be PE when their blood pressure was found to be at least 140/90 mm Hg in two different time intervals of 6 h apart. In addition, hypertension should have been associated with excretion of more than 300 mg urinary protein per 24 h. Patients with chronic hypertension, diabetes mellitus, and renal, and other systemic illnesses were excluded from the study. Normotensive controls were selected from the prenatal clinic and admitted to the study at the time of delivery.

Materials

Hanks’ balanced salt solution (HBSS), DMEM and DMEM-F12, fetal calf serum, penicillin-streptomycin mixture, and Fungizone were obtained from Life Technologies, Inc. (Grand Island, NY). Percoll, 8-bromo adenosine 3'5'-cAMP (8-Br-cAMP), deoxyribonuclease I, BSA, trypsin, and glutamine were purchased from Sigma (St. Louis, MO). All solvents (HPLC grade) were obtained from Merck \|[amp ]\| Co., Inc. (Darmstadt Germany). Unlabeled authentic 25(OH)D₃ 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; specific activity, 30 Ci/mmol) was purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). Human chorionic gonadotrophin (hCG) RIA was kindly provided by NIDDK (Rockville, MD). Human embryonic kidney cells (HEK-293) were purchased from ATCC (CRL-1573; Microbix Biosystems, Ontario, Canada). All other reagents were of analytical grade.

Tissue preparation and cell culture

The isolation and culture of cytotrophoblasts was performed as described by Kliman et al. (11). Briefly, term placentas (35–42 wk of gestation) were obtained from NT and PE women. Tissues were brought immediately to the laboratory, where several cotyledons were removed and rinsed thoroughly in 0.9% NaCl at room temperature. 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 mM 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 mM HEPES and 25 mM 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 (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 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml Fungizone, and 20% heat-inactivated fetal calf serum; plated in 35-mm culture dishes (Nunc, Roskilde, Denmark); and incubated in humidified 5% CO₂-95% air at 37 C. Unless otherwise indicated, after 2 d in culture, cells were incubated in serum-free DMEM-F12 with low calcium and phosphate concentrations.

Daily, the morphological aspects of cell cultures were examined. The concentration of hCG in the culture media was measured, as previously described (7), by specific RIA using antibody anti-hCG-H80, at a final working dilution of 1:150,000. This antibody exhibits 1.2% and 3.2% cross-reactivity 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 less than 10% and less than 6%, respectively. Total protein content of cell cultures was measured by the method of Bradford (12), in a 30-µl aliquot per duplicate, using BSA as standard.

Activity of 1-hydroxylase in cultured human syncytiotrophoblast cells

The ability of syncytiotrophoblast cells to convert 25(OH)D₃ into 1,25(OH)₂D₃ was studied individually in 8 placentas from PE women and 10 placentas from NT subjects, as previously described (7, 10). Briefly, on the third day of culture, medium was changed, and cells were incubated in 2 ml serum-free medium (DMEM-F12) in the presence of [³H]25(OH)D₃, at a final concentration of 3 nM, during 60 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 1-N NaOH. Vitamin D₃ metabolites were extracted from the medium with an additional 3 ml methanol, followed by 4 ml chloroform. The chloroform phase was dried down under N₂, and lipidic extracts were redissolved in chromatographic solvent. Authentic unlabeled 1,25(OH)₂D₃ was used as elution marker, and samples were separated on an HPLC fitted with a photodiode array detector (Model 996; Waters, Milford, MA), using an ultrasphere Si, 5 µm, 4.6 x 250-mm column (Beckman, Palo Alto, CA). A second straight phase HPLC was used to finally separate the vitamin D₃ metabolites (10, 13). The conversion of [³H]25(OH)D₃ into putative [³H]1,25(OH)₂D₃ was determined by estimating the percentage of radioactivity coeluting with authentic unlabeled 1,25(OH)₂D₃ after the 2 successive HPLC’s. Results were expressed as femtomoles per milligram protein.

cDNA synthesis and PCR amplifications

Total RNA, isolated from cultured syncytiotrophoblast cells (14), was used as template for cDNA synthesis, using the SuperScript II preamplification system (Life Technologies, Inc.). PCR amplifications were then performed with Taq polymerase using the following sense and antisense primers: 1-hydroxylase, (5'-GTTGCTATTGGCGGGAGTGGAC-3' and 5'-GTGACACAGAGTGACCAGCATAT-3'); IGF-I, (5'-TCACATCGGCCTCATAATACC-3' and 5'-AAATAAAAGCCCCTGTCTCCA-3'); and hCG, (5'-CGCACCAAGGATGGAGA-3' and 5'-GCCTTTATTGTGGAGGA-3'), which yielded a 298-bp, 229-bp, and 494-bp RT-PCR product, respectively. Normalization was performed by the amplification of cyclophilin mRNA (CF) with the following sense and antisense primers: 5'-CCCCACCGTGTTCTTCGACAT-3' and 5'-AGGTCCTTACCGTTCTGGTCG-3', which yielded a 453-bp RT-PCR product. Incubations, in the absence of reverse transcriptase, were used as controls for the RT-PCR reactions. The PCR products were resolved on agarose gels, blotted onto nylon membranes (15) and hybridized with human 1-hydroxylase or IGF-I cDNAs nested probes (183 bp and 66 bp, respectively) radiolabeled with [³²P]-deoxy-CTP by random priming. The probe for 1-hydroxylase was obtained from HEK-293 cells as previously described (7). The IGF-I and cyclophilin nested probes were generated from human placental tissue by RT-PCR using the following primers: IGF-I (5'-AGCTCTGCCACGGCTGGACCGGAG-3' and 5'-CACGAACTGAAGAGCATCCACCAG-3') and cyclophilin (5'-CACACGCCATAATGGCACTGGTGG-3' and 5'-AAAGACCACATGCTTGCCATCCAGC-3') for sense and antisense primers, respectively. In the case of the ß-subunit of hCG mRNA, the cDNA probe was obtained using the primers for PCR amplifications. The probes were sequenced for identity by ABI PRISM Dye Terminator Cycle Sequencing Kit (Perkin-Elmer Corp., Foster City, CA), as previously described (7).

For Northern blots, 30 µg total cellular RNA was size-fractionated on a 1.2% formaldehyde-agarose gel. After electrophoresis, RNA was transferred onto nylon membranes by capillary diffusion, fixed by UV cross-linking, and probed with the corresponding [³²P]-deoxy-CTP-labeled cDNA. After prehybridization for 1 h at 68 C, the radioactive probe was added and hybridized in 0.25 M Na₂HPO₄ and 7% sodium dodecyl sulfate during 24 h at 68 C.

Statistical analysis

The area under the curve (AUC) of hCG secretion in culture media was calculated by the trapezoid method, with the aid of a computer program (SigmaStat; Jandel Scientific Software, Chicago, IL). Statistical significance among comparisons was established using Student’s t test. A P value 0.05 was considered statistically significant

Results

Functional trophoblast cell differentiation

Microscopic examination of placental cultures from both NT and PE women showed, after 3 d of culture, the presence of cell aggregates containing multiple nuclei with very little, if any, single mononuclear cells. Cultures from PE placentas were microscopically indistinguishable from those from NT pregnancies. In all cases, syncytiotrophoblasts appeared not isolated, but forming a network structure of multinucleated cells with clusters containing an average of more than 10 nuclei. Figure 1 shows the AUC of hCG released from NT and PE trophoblasts during the first 72 h of culture. As depicted, both NT and PE placentas released similar amounts of hCG during the 72-h period, either in the absence or presence of 8-Br-cAMP. In both groups, addition of the cyclic nucleotide analog significantly increased the amount of hCG in the culture media without significant differences between them. Figure 2 shows the temporal expression pattern of hCG mRNA isolated from NT and PE nonstimulated trophoblast cells at different days from plating (d 1–4). Total RNA was extracted from cultured cells and subjected to RT-PCR, using specific primers, as outlined under Subjects and Methods. From the Southern blot analysis, a temporal pattern for the amount of hCG mRNA was demonstrated that closely corresponded to the temporal pattern for released hormone (Fig. 1). The relative abundance of hCG mRNA (Fig. 2A) was obtained by normalizing the 494-bp band intensity (Fig. 2B) with that generated for the constitutive gene cyclophilin (Fig. 2C).

View larger version (13K): [in this window] [in a new window]   Figure 1. hCG secretion by NT and PE cultured syncytiotrophoblasts in the absence (-) or presence (+) of 8-Br-cAMP. Bars, AUC of hCG (mean ± SE.), released during 72 h, of 10 cultured placentas, respectively. *, P < 0.001 vs. without 8-Br-cAMP.

View larger version (38K): [in this window] [in a new window]   Figure 2. Temporal pattern of expression of ß-hCG mRNA in syncytiotrophoblast cells from NT and PE placentas. Daily, total RNA was obtained from cultured cells and subjected to RT-PCR as described in Subjects and Methods. The Southern blot was probed with ß-hCG cDNA (B). Control RT-PCR amplifications in the same samples, using cyclophilin, are shown (C). Normalization of relative optical densities of RT-PCR products of ß-hCG and cyclophilin are shown in A.

To demonstrate the ability of PE syncytiotrophoblasts in culture to synthesize 1,25(OH)₂D₃, cells at the third day of culture were incubated in the presence of [³H]25(OH)D₃ during 1 h. As shown in Fig. 3A, cells from control placentas (n = 10), tested at the third day of culture, actively synthesized 1,25(OH)₂D₃ from the labeled precursor (215 ± 114 fmol/mg protein). By contrast, only one tenth the activity observed in control cells was obtained when syncytiotrophoblasts isolated from PE placentas (n = 8) were incubated under identical conditions (19 ± 11 fmol/mg protein; P < 0.001 vs. control). In all instances, cultures of syncytiotrophoblast cells isolated from PE placentas released hCG nonsignificantly different from control cultures.

View larger version (12K): [in this window] [in a new window]   Figure 3. A, [3H]1,25(OH)2D3 production by syncytiotrophoblast cells cultured from NT and PE placentas. At 72 h after plating, cells were incubated for 1 h in the presence of 3 nM [3H]25(OH)D3, and the conversion products were separated by two-step straight-phase HPLC. Each bar represents the mean ± SD of 10 NT and 8 PE placentas, respectively. *, P < 0.001 vs. NT. B, Effects of IGF-I on [3H]1,25(OH)2D3 production by NT and PE syncytiotrophoblast cultures. Cells were incubated as described above but in the presence of 6.5 nM IGF-I (+) or the vehicle alone (-). *, P < 0.01 vs. control.

To determine whether the low basal and IGF-I-stimulated metabolic conversion of [³H]25(OH)D₃ into [³H]1,25(OH)₂D₃ in cultured syncytiotrophoblasts from PE placentas was a result of an altered expression and/or regulation of placental 1-hydroxylase, the expression of this enzyme was investigated by RT-PCR/Southern blot and Northern blots with and without the addition of IGF-I. 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. Figure 4 shows a representative Southern blot of RT-PCR products of 1-hydroxylase mRNA isolated from 72-h cultured syncytiotrophoblasts. A reduction in the relative abundance of 1-hydroxylase mRNA (Fig. 4B), after normalizing with the band obtained for the constitutive gene cyclophilin (Fig. 4C), was observed in PE placentas, compared with controls (Fig. 4A). Similar results were obtained by Northern blots of total RNA extracted from fresh placental tissue (data not shown). The effects of preincubation with 6.5 nM IGF-I on 1- hydroxylase expression were studied by Northern blots in cultured NT and PE syncytiotrophoblast cells. Three-day cultures were treated with IGF-I or vehicle alone for 24 h. As shown in Fig. 5, a 2.5-kb 1-hydroxylase transcript was detected at low levels in untreated cells, with expression being even lower in cells from PE placentas (Fig. 5A). IGF-I treatment increased the transcript levels in cultures from both NT and PE placentas; however, PE cultures showed a significantly lower transcriptional response to IGF-I.

View larger version (36K): [in this window] [in a new window]   Figure 4. Expression of 1-hydroxylase mRNA (CYP27B1) in cultured syncytiotrophoblast cells from NT and PE placentas. Total RNA was obtained from 3-d cultured cells and then subjected to RT-PCR and Southern blot analysis using a specific 1-hydroxylase cDNA probe (B) and cyclophilin probe (C), respectively. Normalization of relative optical densities of RT-PCR products of 1-hydroxylase and cyclophilin is shown in A.

View larger version (41K): [in this window] [in a new window]   Figure 5. Effects of IGF-I on 1-hydroxylase gene expression in NT and PE syncytiotrophoblast cultures. Three-day cultures were incubated as described above, in the absence (-) or presence (+) of IGF-I. After 4 h incubation, total RNA was extracted and hybridized with the [32P]-labeled 1-hydroxylase cDNA probe (B). 28S ribosomal RNA was used to assess equal loading of placental RNA (C) and for normalization of 1-hydroxylase mRNA levels (A). *, P < 0.05 vs. control.

During differentiation of NT and PE cytotrophoblasts, total RNA was extracted at different times of plating and was used as template to generate a RT-PCR IGF-I 229-bp product. The results in Fig. 6 show that the level of IGF-I gene expression was not different in both NT and PE cultures; suggesting that IGF-I might not be the cause for lower 1-hydroxylase activity in PE cell cultures. In addition, an increasing expression pattern of IGF-I gene was observed as trophoblast differentiation process progressed, and it was similar in NT and PE cultures. The relative abundance of IGF-I mRNA was established by normalizing the 229-bp band intensity with cyclophilin RT-PCR product (Fig. 6, C and A, respectively).

View larger version (46K): [in this window] [in a new window]   Figure 6. Temporal expression of IGF-I mRNA in cultured syncytiotrophoblast cells from NT and PE placentas. Daily total RNA was obtained from cultured cells and then subjected to RT-PCR and Southern blot analysis using specific IGF-I probe (B) and cyclophilin (C), respectively. Normalization of relative optical densities of RT-PCR products of IGF-I and cyclophilin is shown in A.

Increase in maternal serum levels of 1,25(OH)₂D₃ has been considered as one of the mechanisms by which calcium absorption is enhanced during pregnancy (16). Although the factors involved in regulating maternal 1,25(OH)₂D₃ serum levels remains largely unknown, we have presented evidence that human placenta synthesized the active metabolite of vitamin D₃ (7, 10) through gene expression and activation of the cytochrome P₄₅₀ 1-hydroxylase. It is also well known that circulating levels of 1,25(OH)₂D₃ are significantly lower in PE women than in normotensive and chronically hypertensive pregnant subjects (3, 4, 6, 17). In addition, these observations strongly raise the possibility that low levels of 1,25(OH)₂D₃ in PE could be the result of a deficient production of this active metabolite by the placenta.

The results presented herein demonstrated a clear significant difference in the ability of cultured syncytiotrophoblast cells isolated from PE placentas to convert [³H]25(OH)D₃ into [³H]1,25(OH)₂D₃. In all instances, cultures from NT placentas produced significantly higher proportions of 1,25(OH)₂D₃ than PE cultures, regardless of the time at which syncytiotrophoblast cells were tested. These results agreed also with those, herein presented, on the relative abundance of 1-hydroxylase mRNA in the same cultured cells. The finding that PE placentas expressed less 1-hydroxylase mRNA and 1-hydroxylase activity than NT controls may indicate a specific alteration in placental ability to synthesize adequate amounts of 1-hydroxylase. Our results showing that trophoblasts from PE pregnancies were able to normally differentiate into syncytiotrophoblasts and produced hCG in response to 8-Br-cAMP, in a manner similar to that of control cells, argued against an overall placental restricted metabolic capacity or a deficient cell differentiation process as factors responsible for low 1-hydroxylase gene expression and enzymatic activity. This observation is in line with previous studies indicating that invasive trophoblast is more likely to be altered in PE than endocrine syncytiotrophoblast (18).

It has been an intriguing possibility that placental production of 1,25(OH)₂D₃ is altered in PE. Until now, there was no way to differentiate, by the current available methodology, between 1,25(OH)₂D₃ produced by the kidney vs. that produced by the placenta. In this study, we suggest that the etiology of vitamin D metabolic alterations seen in PE may reside, in part, in the placenta and is attributable to a deficient production of 1-hydroxylase. The results presented herein clearly demonstrated that synthesis and activity of 1-hydroxylase were indeed significantly restricted in cultured human syncytiotrophoblasts obtained from PE pregnancies. In this regard, PTH and 1,25(OH)₂D₃ are two of the major physiological factors among those involved in regulating 1,25(OH)₂D₃ production (19); and, until now, the molecular regulatory mechanisms of this enzyme have been partially clarified (20, 21). Interestingly, studies in humans have clearly indicated that both 1,25(OH)₂D₃ and IGF-I serum concentrations are significantly lower in PE than in NT pregnancies (6). These studies, linked to others indicating IGF-I as a unique calcium-dependent stimulator of renal (9) and probably placental 1,25(OH)₂D₃ production (10), prompted us to further investigate whether this factor was involved in the low 1-hydroxylase activity in PE placentas. Although the role of IGF-II on placental 1-hydroxylase cannot be ruled out, its relative abundance in the intermediate trophoblast, which seemed to increase as the cells invade into the maternal decidua (22), suggests that IGF-II would be involved rather in the process of invasion, growth, and differentiation of the trophoblast.

Expression of IGF mRNAs has been previously demonstrated in human placenta (23, 24). Both IGF-I and IGF-II mRNAs have similar distribution, but IGF-II mRNA is more abundant in placentas obtained at all gestational ages. These data, taken together with those demonstrating the presence of IGF-I receptors on placental membranes (25, 26), suggest an autocrine/paracrine mechanism of IGF-I, to regulate placental growth and metabolism, including 1-hydroxylase expression and/or activity. Although, in this study, the role of other 1-hydroxylase regulatory factors in the placenta cannot be ruled out, our results demonstrating that IGF-I expression in PE cultures was similar to that observed in NT cells, suggest that low 1-hydroxylase expression and activity in PE were probably not the result of alterations in IGF-I locally produced at the placental level. However, an increase in the concentrations of IGF binding protein-1 in maternal serum and at the decidual-trophoblast interface, as described in PE (22), may decrease the bioavailability and biological activity of both systemic and locally produced IGF-I at the placental level. In addition, altered IGF-I and insulin action in erythrocytes of PE patients have been described (27), which may also be of relevance in many of the actions mediated by this growth factor in both maternal and fetal tissues. These observations agreed with our results on the effects of IGF-I on 1-hydroxylase gene expression and enzyme activity.

On the other hand, it is also plausible that liver, rather than placental IGF-I, could be involved in regulating 1-hydroxylase in the placenta, because variations of IGF-I in maternal serum during pregnancy parallel the changes in IGF-I mRNA in the liver (28). In addition, NT expression of IGF-I mRNA in placental cultures from PE pregnancies, during the time of syncytiotrophoblasts forming was coincident with changes in cell morphology and hormone secretion accompany trophoblast differentiation.

It is well known that IGF-I is primarily regulated by pituitary GH (29); however, during pregnancy, GH synthesis by the pituitary is inhibited, placental GH being the primary GH species in the maternal circulation (30). In PE, where placental invasion is compromised, it is possible that placental GH synthesis is also affected, thereby resulting in decreased liver IGF-I production. The consequence of reduced placental 1,25(OH)₂D₃, most probably as the result of the low plasma levels of IGF-I in PE, is still unclear. However, the presence of 1,25(OH)₂D₃ receptors in the human placenta (31) suggests the involvement of 1,25(OH)₂D₃ in the process of transport of calcium across the placenta. Indeed, the placenta is able to transport calcium actively even in the absence of fetal parathyroid glands (32), a mechanism that is most probably regulated by the locally produced 1,25-(OH)₂D₃ or other factors, such as PTHrP (33).

In summary, our results gave further support suggesting the placenta as a contributor of 1,25-(OH)₂D₃ during pregnancy. In addition, cultured syncytiotrophoblast cells from PE placentas expressed less 1-hydroxylase mRNA and 1-hydroxylase enzymatic activity than NT placental cells. These results may provide an explanation for a number of metabolic alterations in PE associated with changes in 1,25-(OH)₂D₃ production. Further studies on the elucidation of the mechanisms involved in the regulation of extrarenal 1,25-(OH)₂D₃ production will be of importance to our understanding of the tissue-specific functions of 1-hydroxylase.

Acknowledgments

We acknowledge and give thanks to the National Hormone and Pituitary Program, the National Institute of Diabetes and Digestive and Kidney Diseases, the National Institute of Child Health and Human Development, and the United States Department of Agriculture for hCG RIA reagents, and to F. Hoffmann-La Roche Ltd. for 25(OH)D₃ and 1,25(OH)₂D₃ standards. We acknowledge Biologist Irene Sánchez for her valuable technical assistance.

Footnotes

This work was supported by grants from The Special Program for Research, Development and Research Training in Human Reproduction of the World Health Organization (Geneva, Switzerland) and The National Council of Science and Technology (CONACyT, México). C.A. was the recipient of a fellowship from Programa Latinoamericano de Capacitación e Investigación en Reproducción Humana (PLACIRH, México) and Programa Regional en Salud Sexual y Reproductiva para America Latina y el Caribe (PROGRESAR, Chile).

Abbreviations: AUC, Area under the curve; 8-Br-cAMP, 8-bromo adenosine 3'5'-cAMP; HBSS, Hanks’ balanced salt solution; hCG, human chorionic gonadotrophin; 1-hydroxylase, 25-hydroxyvitamin D-1-hydroxylase; NT, normal; [³H]25(OH)D₃, 25-hydroxy-[26,27-methyl-³H]cholecalciferol_; 1,25(OH)₂D₃, 1,25-dihydroxyvitamin D₃; PE, preeclampsia.

Received January 16, 2002.

Accepted April 18, 2002.

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