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Am J Physiol Endocrinol Metab 290: E135-E142, 2006. First published September 6, 2005; doi:10.1152/ajpendo.00356.2005
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Cadmium reduces 11{beta}-hydroxysteroid dehydrogenase type 2 activity and expression in human placental trophoblast cells

Kaiping Yang, Laura Julan, Fran Rubio, Anju Sharma, and Haiyan Guan

Canadian Institutes of Health Research Group in Fetal and Neonatal Health and Development, Children's Health Research Institute and Lawson Health Research Institute, Departments of Obstetrics and Gynaecology and Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada

Submitted 2 August 2005 ; accepted in final form 31 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cadmium, a common environmental pollutant and a major constituent of tobacco smoke, has been identified as a new class of endocrine disruptors with a wide range of detrimental effects on mammalian reproduction. During human pregnancy, maternal cadmium exposure, via the environment and/or cigarette smoking, leads to fetal growth restriction (FGR), but the underlying mechanisms are unknown. Although a substantial amount of evidence suggests that cadmium may affect fetal growth indirectly via the placenta, the molecular targets remain to be identified. Given that reduced placental 11{beta}-hydroxysteroid dehydrogenase type 2 (11{beta}-HSD2, encoded by HSD11B2 gene) is causally linked to FGR, the present study was undertaken to examine the hypothesis that cadmium induces FGR in part by targeting placental HSD11B2. Using cultured human trophoblast cells as a model system, we showed that cadmium exposure resulted in a time- and concentration-dependent decrease in 11{beta}-HSD2 activity, such that an 80% reduction was observed after 24-h treatment at 1 µM. It also led to a similar decrease in levels of 11{beta}-HSD2 protein and mRNA, suggesting that cadmium reduced 11{beta}-HSD2 expression. Furthermore, cadmium diminished HSD11B2 promoter activity, indicative of repression of HSD11B2 gene transcription. In addition, the effect of cadmium was highly specific, in that other divalent metals (Zn2+, Mg2+, and Mn2+) as well as nicotine and cotinine (a major metabolite of nicotine) did not alter 11{beta}-HSD2 activity. Taken together, these findings demonstrate that cadmium reduces human placental 11{beta}-HSD2 expression and activity by suppressing HSD11B2 gene transcription. Thus the present study identifies placental 11{beta}-HSD2 as a novel molecular target of cadmium. It also reveals a molecular mechanism by which this endocrine disruptor may affect human placental function and, consequently, fetal growth and development.

glucocorticoid; placenta; fetal growth restriction; environmental toxin

CADMIUM IS WIDELY DISPERSED in the environment mainly as result of pollution from a variety of sources, including mining, smelting, fossil fuel combustion, batteries, paints, and plastics (30, 80). Additionally, one important source of this heavy metal in the environment is tobacco smoke (82). Cadmium has been classified as a human carcinogen by the International Agency for Research on Cancer (76). It has also been identified recently as a new class of endocrine disruptors, and exposure to this metal is linked to a wide range of detrimental effects on mammalian reproduction (29). Cadmium exposure during human pregnancy is associated with low birth weight, spontaneous abortion, and premature delivery (29, 53).

Previous studies in both animals and humans demonstrate that cadmium accumulates in high concentrations in the placenta, and very little crosses the placenta into the fetus (29). This suggests that the placenta is a primary target for this metal during mammalian pregnancy. Indeed, placental structural abnormalities were observed in women who smoked (9, 46, 74) and in various animals exposed to cadmium during pregnancy (16). In perfused human placentas in vitro, cadmium exposure caused decreased secretion of human chorionic gonadotropin (78), a peptide hormone vital to early pregnancy. Furthermore, progesterone levels were inversely correlated with levels of cadmium in placentas of women who smoked during pregnancy (61). There is also direct evidence that cadmium reduces the biosynthesis of progesterone in cultured human trophoblast cells (32, 33, 38). Given that progesterone plays a key role in maintaining myometrial quiescence (11, 13), the cadmium-induced downregulation of placental progesterone synthesis may provide a mechanistic basis for the association between smoking and premature delivery. Although fetal hypoxia, disruption of normal fetoplacental zinc homeostasis, and reduced uteroplacental blood flow have been proposed as potential mechanisms contributing to cadmium-induced fetal growth restriction (FGR) (29, 53, 81), the precise molecular targets of cadmium in the placenta are poorly defined.

Recently, the placental 11{beta}-hydroxysteroid dehydrogenase type 2 (11{beta}-HSD2; encoded by the HSD11B2 gene) enzyme has emerged as a key player in controlling fetal development for the following reasons (8, 79). First, owing to its localization to the syncytiotrophoblast layer of the human placenta (43, 60), the site of maternal-fetal exchange, placental 11{beta}-HSD2 serves as a functional barrier to protect the fetus from exposure to high levels of maternal glucocorticoid by converting maternal cortisol to its inactive metabolite cortisone. Second, placental 11{beta}-HSD2 activity is positively correlated with birth weight (56, 71). Third, FGR is a characteristic feature of 11{beta}-HSD2 deficiency resulting from mutations in the HSD11B2 gene in humans (39). Importantly, reduced placental 11{beta}-HSD2 (both expression and activity) is associated with pregnancies complicated with FGR (17, 35, 54, 56, 68, 69). Therefore, we hypothesized that cadmium induces FGR in part by targeting the placental HSD11B2 gene. In the present study, we tested this hypothesis utilizing cultured human placental trophoblast cells as a model system. We present the first evidence that cadmium, but not nicotine or cotinine (a major metabolite of nicotine), suppresses HSD11B2 gene transcription, leading to reduced 11{beta}-HSD2 expression and activity in cultured human trophoblast cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Overview. In this study, we utilized our established primary human trophoblast cells as a model system in which to sequentially study the effects of cadmium on placental 11{beta}-HSD2 activity, 11{beta}-HSD2 protein, 11{beta}-HSD2 mRNA, and HSD11B2 promoter activity. The primary focus of our study was on placental 11{beta}-HSD2 activity, because it represents the most important biological readout. First, we determined whether the effect of cadmium on placental 11{beta}-HSD2 activity was time and concentration dependent by treating trophoblast cells with CdCl2 for different times and at various concentrations. Second, we treated trophoblast cells with three other divalent metals, ZnCl2, MgCl2, and MnCl2, to determine whether the inhibitory effect of CdCl2 on placental 11{beta}-HSD2 activity was specific to cadmium. Third, we incubated trophoblast cells with nicotine and cotinine (a major metabolite of nicotine), two well-known constituents of tobacco smoke besides cadmium, to determine whether these two compounds had similar effects on placental 11{beta}-HSD2 activity. Fourth, we determined sequentially whether cadmium reduced expression of 11{beta}-HSD2 protein and mRNA. Furthermore, we examined the effect of cadmium on the mRNA expression of heme oxygenase-1 (HO-1; a well-known inducible target of cadmium) to provide evidence that cadmium did not cause global downregulation of gene expression in trophoblast cells. In the last experiment, we performed transient transfection experiments using a luciferase reporter assay to determine whether the effect of cadmium on placental HSD11B2 occured at the transcriptional level.

Placental trophoblast cell cultures. Placental trophoblast cells were isolated using a modification of the method of Kliman et al. (40), as described (28). Ethics approval for procurement of human placentas was obtained from the University of Western Ontario Ethics Board for Health Sciences Research Involving Human Subjects. Briefly, human placentas were obtained from uncomplicated pregnancies (i.e., without maternal or fetal diseases or maternal substance abuse, including smoking) at term after elective cesarean section. Villous tissues were dissected free from fetal membranes and blood vessels, rinsed in 0.9% NaCl2, and digested with 0.125% trypsin and 0.02% deoxyribonuclease-I (Sigma) in DMEM containing 0.05% streptomycin and gentamicin (Invitrogen) three times for 30 min each. The placental cells were loaded onto a 5–70% Percoll gradient at step increments of 5% Percoll, and centrifuged at 2,500 g for 20 min to separate different cell types. Cytotrophoblasts between the density markers of 1.049 and 1.062 g/ml were collected and plated in either 24-well plates (for enzyme activity assay) or 35-mm dishes (for Western blot analysis) at a density of 1.35 x 106 cells/ml in M199 containing 10% FCS (Invitrogen). The cells were maintained at 37°C in humidified 5% CO2-95% air (20% O2) for 48 h. We have shown previously (28) that the isolated cytotrophoblasts will differentiate into syncytiotrophoblasts over 48 h of culture under conditions of the present study. After the end of 48 h, the trophoblast cells (in triplicate) were treated for 24 h (or as indicated otherwise) with various compounds in the medium containing 2% FCS. Controls, also in triplicate, received equivalent volume of vehicle.

Assay of 11{beta}-HSD2 activity: radiometric conversion assay. The level of 11{beta}-HSD2 activity in intact cells at various time points and following different treatment regimens was determined by a radiometric conversion assay, as described previously (28). Briefly, the cells were incubated for 1 h at 37°C in serum-free medium containing ~50,000 cpm [3H]cortisol and 100 nM unlabeled cortisol. At the end of incubation, the medium was collected and steroids were extracted. The extracts were dried and the residues resuspended. A fraction of the resuspension was spotted on a TLC plate that was developed in chloroform-methanol (9:1, vol/vol). The bands containing the labeled cortisol and cortisone were identified by UV light of the cold carriers, cut out into scintillation vials, and counted in Scintisafe Econo 1 (Fisher Scientific). The rate of cortisol-to-cortisone conversion was calculated, and the blank values (defined as the amount of conversion in the absence of cells) were subtracted and expressed as percentage of control. Results are shown as means ± SE.

Protein extraction and Western blot analysis. Western blot analysis was used to determine changes in the level of 11{beta}-HSD2 protein. After treatment with cadmium, both control and cadmium-treated cells were lysed with cold lysis buffer (100 mM NaCl, 50 mM NaF, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.1 mM PMSF, 1 mM orthovanadate, and 50 mM Tris·HCl, pH 7.5) for 5 min at room temperature. The total cell lysates were collected with a cell scraper, vortexed vigorously, and centrifuged at 10,000 g for 20 min at 4°C. The supernatant was collected, and the protein content was determined by the Bradford method using a protein assay kit (Bio-Rad Laboratories, Missasauga, ON, Canada) with BSA as a standard.

Western blot analysis was conducted as described previously (28). Briefly, 30 µg of the protein extracts were subjected to a standard 12% SDS-PAGE. After electrophoresis, proteins were transferred to nitrocellulose using a Bio-Rad Mini Transfer Apparatus. The 11{beta}-HSD2 protein was detected on the nitrocellulose filter by use of an ECL Western Blotting Analysis System (Pharmacia) following the manufacturer's instructions. Briefly, the nitrocellulose filter was blocked overnight at 4°C with 10% Blotto in TTBS (0.1% Tween-20 in TBS), and incubated with primary antibody (HUH23; 0.25 µg/ml in TTBS) for 1 h at room temperature. The primary antibody was a polyclonal rabbit anti-human 11{beta}-HSD2 antibody (a generous gift from Dr. Z. Krozowski) (43). After three 5-min washes with TTBS, the filter was incubated with horseradish peroxidase-labeled second antibody (1:5,000 dilution) and developed in ECL detection reagents. The filter was then exposed to X-ray film (Eastman Kodak, Rochester, NY) for 1–5 min. Densitometry was performed on the radiographs, and the level of 11{beta}-HSD2 protein was expressed as percentage of controls.

Assessment of 11{beta}-HSD2 mRNA and HO-1 mRNA: real-time quantitative RT-PCR. To determine whether cadmium-induced concentration-dependent decreases in 11{beta}-HSD2 activity were associated with corresponding reductions in 11{beta}-HSD2 mRNA levels, trophoblast cells were treated for 24 h with various concentrations of CdCl2, and the relative abundance of 11{beta}-HSD2 mRNA was assessed by two-step real-time quantitative RT-PCR (qRT-PCR), as described previously (34). Given that HO-1 is a well-known inducible target of cadmium (51), changes in the level of HO-1 mRNA in cultured trophoblast cells following CdCl2 treatment at various concentrations were also determined as described below.

Briefly, total RNA was extracted from cultured cells using an RNeasy Mini Kit (QIAGEN, Mississauga, ON, Canada) coupled with on-column DNase digestion with the RNase-Free DNase Set (QIAGEN) according to the manufacturer's instructions. One-half microgram of total RNA was reverse transcribed in a total volume of 20 µl using the High Capacity Complimentary Deoxyribonucleic acid (cDNA) Archive Kit(Applied Biosystems, Forest City, CA) following the manufacturer's instructions. For every RT reaction set, one RNA sample was set up without RT enzyme to provide a negative control. Gene transcript levels of 18S rRNA (housekeeping gene) and 11{beta}-HSD2 were quantified separately by TaqMan assays using the TaqMan Universal PCR Master Mix (Applied Biosystems) and the universal thermal cycling parameters (2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C) on an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Levels of 18S rRNA were assessed using TaqMan Ribosomal RNA Control Reagents (Applied Biosystems), and those of 11{beta}-HSD2 mRNA using custom-designed TaqMan assays. Primers (60 nM each) and TaqMan MGB probe (200 nM) for human 11{beta}-HSD2 were designed using the Primer Express Software (Applied Biosystems; Table 1), and their optimal concentrations were determined following guidelines developed for Sequence Detection Systems by Applied Biosystems.


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  		Table 1. Human 11{beta}-HSD2 TaqMan assay primers and probe and human HO-1 SYBR Green I assay primers

 
The level of HO-1 mRNA was determined by a custom-designed SYBR Green I chemistry-based assay (27). Briefly, primers (300 nM each) for human HO-1 were designed with the same Primer Express Software (Applied Biosystems; Table 1), and the optimal concentrations were determined empirically. The SYBR Green I assay was performed with the SYBR Green PCR Master Mix (Applied Biosystems) and a modified universal thermal cycling condition (2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 10 s each at 95, 60, and 72°C) with the standard disassociation/melting parameters (15 s each at 95, 60, and 95°C) on the ABI Prism 7900HT Sequence Detection System. The specificity of the SYBR Green I assay was verified by performing a melting-curve analysis (single peak with a Tm of 83.7°C) and by subsequent sequencing of the PCR products.

Levels of 18S rRNA, 11{beta}-HSD2 mRNA, and HO-1 mRNA in each RNA sample were quantified by the relative standard curve method (Applied Biosystems). Briefly, standard curves for 18S rRNA, 11{beta}-HSD2 mRNA and HO-1 mRNA were generated by performing a dilution series of the untreated control cDNA. For each RNA sample, the relative amount of 18S rRNA, 11{beta}-HSD2 mRNA and HO-1 mRNA was obtained, and the ratio of 11{beta}-HSD2 mRNA to 18S rRNA as well as the ratio of HO-1 mRNA to 18S rRNA were calculated. For each experiment, the amount of 11{beta}-HSD2 mRNA or HO-1 mRNA at any given concentration of CdCl2 is expressed relative to the amount of transcript present in the untreated control.

Transient transfection and reporter gene assay. To determine whether cadmium altered the rate of HSD11B2 gene transcription, a standard reporter gene assay was performed as described (34). Briefly, the isolated trophoblast cells were plated on 24-well plates and cultured under standard conditions for 48 h. The cells were then cotransfected with 0.8 µg/well of the pGL3-HSD11B2P+330bp and 0.2 µg/well of a cytomegalovirus promoter (pCMV) {beta}-galactosidase plasmid (Promega, Madison, WI), or with 0.8 µg/well pGL3-Basic and 0.2 µg/well pCMV-{beta}-Gal (negative control). All transfections were carried out in serum-free M199 for 1 h using Transfast Transfection Reagent (Promega) at a ratio of 2:1 (transfection reagent to DNA) according to the manufacturer's instructions. At the end of transfection, cells were gently overlaid with fresh medium containing 10% serum and incubated for 4 h. The medium was then replaced with fresh medium containing 2% serum, and the cells were treated with 1.0 µM CdCl2 for 24 h. At the end of treatment, luciferase and galactosidase activities were analyzed using the Luciferase Assay System (Promega) and the {beta}-Galactosidase Enzyme Assay System (Promega), respectively. Luciferase activity was measured using a Lumat LB 9507 luminometer (EG&G Berthold, Bad Wildbad, Germany) and normalized against {beta}-galactosidase activity. Each transfection was performed in triplicate, and a total of three to five independent experiments were carried out.

Reagents and supplies. [1,2,6,7-3H(N)]cortisol (80 Ci/mmol) was purchased from DuPont Canada (Markam, ON). Nonradioactive cortisol and cortisone were obtained from Steraloids (Wilton, NH). CdCl2, ZnCl2, MgCl2, MnCl2, nicotine, and cotinine were purchased from Sigma-Aldrich Canada (Oakville, ON, Canada). Polyester-backed thin-layer chromatography (TLC) plates were obtained from Fisher Scientific (Nepean, ON). All solvents used were from VWR Canlab (Mississauga, ON, Canada). Cell culture supplies were obtained from either Invitrogen Life Technologies (Burlington, ON, Canada) or Fisher Scientific. General molecular biology reagents were from Invitrogen or Pharmacia Canada (Baie D'Urte, QC, Canada). Oligonucleotides were synthesized (11{beta}-HSD2 primers) by a Pharmacia Gene Assembler and purified with NAP-50 columns (Pharmacia) according to the manufacturer's instructions or purchased (HO-1 primers) from Sigma Genosys (Oakville, ON, Canada).

Statistical analyses. Results are presented as means ± SE of three to five independent experiments (i.e., tissues from different patients), as indicated. Statistical analyses of 11{beta}-HSD2 activity and mRNA as well as HO-1 mRNA data were performed using one-way ANOVA followed by Tukey's post hoc test. 11{beta}-HSD2 protein and promoter activity data were analyzed by a standard Student's t-test. Significance was set at P < 0.05. Calculations were performed using SPSS software version 9.0 (Chicago, IL).


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of cadmium on 11{beta}-HSD2 activity. To study the effects of cadmium on placental 11{beta}-HSD2 activity, isolated human trophoblast cells were treated with 1.0 µM CdCl2 for different times (3, 6, 12 and 24 h). This treatment resulted in a time-dependent decrease in 11{beta}-HSD2 activity such that a significant reduction (P < 0.05) occurred after 3 h of treatment (Fig. 1A). Furthermore, the cells were also treated for 24 h with various concentrations of CdCl2 (0.25 to 1.0 µM), and this led to a concentration-dependent decrease in 11{beta}-HSD2 activity with a maximal effect at 0.75 µM CdCl2 (20% of control; Fig. 1B).



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  		Fig. 1. Time- and concentration-dependent effects of CdCl2 on 11{beta}-hydroxysteroid dehydrogenase type 2 (11{beta}-HSD2) activity. A: time-dependent effects of CdCl2 on 11{beta}-HSD2 activity. Trophoblast cells were incubated with or without 1.0 µM CdCl2 for various periods of time (3, 6, 12, and 24 h). B: concentration-dependent effects of CdCl2 on 11{beta}-HSD2 activity. Trophoblast cells were treated for 24 h with increasing concentrations of CdCl2 (0.25, 0.50, 0.75, and 1.0 µM). At end of treatment, the level of 11{beta}-HSD2 activity in intact cells was determined by standard radiometric conversion assay, as described in MATERIALS AND METHODS. Each data point is expressed as percentage of control (separate controls without CdCl2 were used for each time point in A), and each bar represents mean ± SE of 4 independent experiments each performed in triplicate (*P < 0.05 vs. control).

 
Effects of other divalent metals on 11{beta}-HSD2 activity. To determine whether decreases in placental 11{beta}-HSD2 activity following treatment with CdCl2 were specific to this metal, we treated trophoblast cells for 24 h with three other divalent metals, ZnCl2, MgCl2, and MnCl2, each at 1.0 µM (corresponding to the maximal effective concentration of CdCl2) and 10 µM concentrations. In contrast to cadmium, none of these divalent metals reduced 11{beta}-HSD2 activity (data not shown), indicating that the effect of cadmium on placental 11{beta}-HSD2 is not a general property of divalent metals.

Effects of nicotine and cotinine on 11{beta}-HSD2 activity. Besides cadmium, nicotine and cotinine (a major metabolite of nicotine) are well-known constituents of tobacco smoke (4). To determine whether these two compounds have similar effects on placental 11{beta}-HSD2 activity, trophoblast cells were treated for 24 h with various concentrations of nicotine and cotinine (0.1, 0.5, and 1.0 mM). In contrast to cadmium, nicotine and cotinine, at concentrations up to 1 mM, did not decrease 11{beta}-HSD2 activity in cultured trophoblast cells (data not shown).

Effects of CdCl2 on 11{beta}-HSD2 protein and mRNA. To determine whether the CdCl2-induced decrease in 11{beta}-HSD2 activity was a result of reduced 11{beta}-HSD2 expression, levels of 11{beta}-HSD2 protein and mRNA were assessed by Western blot analysis and qRT-PCR, respectively. As shown in Fig. 2, treatment of trophoblast cells for 24 h with 1.0 µM CdCl2 led to a profound reduction in levels of 11{beta}-HSD2 protein (25% of control). Similar to its effects on 11{beta}-HSD2 activity, cadmium resulted in a concentration-dependent decrease in the steady-state level of 11{beta}-HSD2 mRNA with a maximal effect at 0.75 µM (10% of control; Fig. 3A).



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  		Fig. 2. Effects of CdCl2 on 11{beta}-HSD2 protein. Trophoblast cells were treated with 1.0 µM CdCl2 for 24 h. At end of treatment, levels of 11{beta}-HSD2 protein were determined by western blot analysis as described in MATERIALS AND METHODS. A: one representative autoradiograph is shown. B: each bar represents mean ± SE of 4 independent experiments (**P < 0.01 vs. control).

 


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  		Fig. 3. Effects of CdCl2 on 11{beta}-HSD2 mRNA and heme oxygenase-1 (HO-1) mRNA. Trophoblast cells were treated with increasing concentrations of CdCl2 (0.25, 0.50, 0.75, and 1.0 µM) for 24 h. At end of treatment, total cellular RNA was isolated, and the steady-state level of 11{beta}-HSD2 mRNA (A) and HO-1 mRNA (B) was assessed by quantitative RT-PCR, as described in MATERIALS AND METHODS. Each data point is expressed as percentage of control, and each bar represents mean ± SE of 4 independent experiments (*P < 0.05 vs. control).

 
Effects of CdCl2 on HO-1 mRNA. To determine whether cadmium causes global downregulation of gene expression in trophoblast cells, the level of HO-1 mRNA (a well-known inducible target of cadmium) was measured following treatment for 24 h with 1 µM CdCl2, by qRT-PCR. As shown in Fig. 3B, cadmium resulted in a concentration-dependent increase in HO-1 mRNA levels. Furthermore, there was no evidence of CdCl2-induced cytotoxicity, as determined by trypan blue exclusion (data not shown). Collectively, these observations suggest that the inhibitory effects of cadmium on placental 11{beta}-HSD2 are specific and cannot be attributed to global downregulation of gene expression resulting from cytotoxicity.

Effects of CdCl2 on HSD11B2 gene transcription. To determine whether the effect of cadmium on placental HSD11B2 occurs at the transcriptional level, transient transfection experiments were performed. Trophoblast cells were cotransfected with a luciferase construct containing a 330-bp 5'-flanking region of the human HSD11B2 gene (Fig. 4A) and pCMV-{beta}-Gal plasmid DNA. The level of luciferase activity was normalized to that of {beta}-galactosidase and used as an indicator of HSD11B2 promoter activity. Treatment with CdCl2 (1.0 µM) resulted in a 75% reduction in HSD11B2 promoter activity (Fig. 4B).



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  		Fig. 4. Effects of CdCl2 on HSD11B2 gene promoter activity. A: schematic representation of the reporter gene construct. Trophoblast cells were transfected with a luciferase (Luc) reporter gene construct driven by the HSD11B2 promoter and pCMV {beta}-galactosidase (B), as described in MATERIALS AND METHODS. Four hours after transfection, cells were treated with 1.0 µM CdCl2 for 24 h. At end of treatment, luciferase and {beta}-galactosidase ({beta}-Gal) activities were determined, and the ratio of luciferase activity to that of {beta}-galactosidase was calculated and expressed as percentage of control. Each bar represents mean ± SE of 4 independent experiments (**P < 0.01 vs. control).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
FGR affects 5–10% of all pregnancies and is a leading cause of perinatal morbidity and mortality (6, 48). Moreover, accumulating evidence suggests that FGR is also a significant risk factor for obesity, type 2 diabetes, and cardiovascular diseases later in life (58). Of all the identified etiological determinants of FGR in developed countries, the most significant is prenatal exposure to cigarette smoking (42, 63). Furthermore, a dose-dependent relationship exists between the reduction in birth weight and the number of cigarettes smoked per day (12). In the United States, maternal smoking is responsible for 20–30% of low-birth-weight infants and 10% of infant deaths (64, 65). In addition, maternal smoking is a leading risk factor for sudden infant death syndrome in FGR infants (14, 67). The growth-inhibiting effect of tobacco smoke is observed not only in cases of heavy maternal smoking (≥10 cigarettes/day) but also in instances of light (<10 cigarettes/day) and even passive smoking (50). Importantly, recent evidence also suggests that there is a transgenerational effect of maternal smoking such that a mother's prenatal exposure to smoke may affect the birth weight of her offspring (55).

Because tobacco smoke contains several thousand chemicals (21, 70), identification of the causative agents of FGR by cigarette smoking has been a formidable task. One component of tobacco smoke found preferentially to accumulate in the placenta, and investigated extensively in both humans and animal models, is cadmium (29, 53). During human pregnancy, maternal exposure to cadmium is associated with low birth weight and an increased incidence of spontaneous abortion (23, 29, 44, 66). In rodents, maternal cadmium administration during pregnancy leads to FGR (3, 36). Although pharmacokinetic studies have demonstrated that the metal does not readily reach the fetus, it accumulates in high concentrations in the placenta (29), suggesting that the placenta is a primary target by which cadmium influences fetal growth and development. Indeed, both structural and functional abnormalities were observed in placentas of humans and rodents exposed to cadmium during pregnancy (9, 16, 46, 74). Proposed mechanisms by which cadmium leads to FGR include reduced uteroplacental blood flow (7, 10, 47) and impaired placental transport function (15, 26, 49, 59). In particular, there is robust evidence that disruption of normal fetoplacental zinc homeostasis is an important mechanism contributing to cadmium-induced FGR (53). However, the molecular targets within the placenta that are responsible for mediating the growth-inhibiting effect of cadmium remain largely unknown.

Given that reduced placental 11{beta}-HSD2 is causally linked to FGR (54), we hypothesized that cadmium induces FGR in part by downregulating placental 11{beta}-HSD2. As a first step in examining this hypothesis, we studied the effects of cadmium on placental 11{beta}-HSD2 by utilizing cultured human trophoblast cells as a model system. We demonstrated that treatment of trophoblast cells with cadmium led to a time- and concentration-dependent decrease in the level of 11{beta}-HSD2 activity. Furthermore, the cadmium-induced decrease in 11{beta}-HSD2 activity was a consequence of reduced 11{beta}-HSD2 expression, as levels of both 11{beta}-HSD2 protein and mRNA were decreased following treatment with the metal.

In theory, a decrease in the steady-state mRNA level of a given gene can be achieved by reducing the rate of gene transcription and/or by decreasing the mRNA stability. To determine whether the cadmium-induced decrease in 11{beta}-HSD2 mRNA was mediated at the level of HSD11B2 gene transcription, we transiently transfected trophoblast cells with a reporter gene construct driven by the proximal HSD11B2 promoter and examined the effect of cadmium on the reporter gene activity. Our results demonstrated that cadmium reduced HSD11B2 promoter activity by 75%, which was similar in magnitude to the reduction in levels of 11{beta}-HSD2 mRNA, suggesting that the inhibitory effect of cadmium on placental 11{beta}-HSD2 activity and expression was mediated primarily at a transcriptional level. Given that the proximal HSD11B2 promoter contains multiple Sp1 binding sites (1, 57) and that cadmium has been shown to reduce Sp1 binding activity in adult rat alveolar epithelial cells (77), it remains possible that cadmium may suppress HSD11B2 gene transcription by interfering with Sp1 activity in human trophoblast cells. Obviously, this contention warrants future study.

Although the signal transduction pathway involved in mediating the effects of cadmium on placental 11{beta}-HSD2 remains to be defined, two pathways may be proposed, and they are currently under investigation in our laboratory. Given that cadmium activates the extracellular signal-regulated kinase (ERK) in a variety of mammalian cells (76) and that the activation of ERK has been shown to decrease 11{beta}-HSD2 activity in the JEG-3 trophoblast cell line (45), the inhibitory effects of cadmium on placental 11{beta}-HSD2 may be mediated by the ERK pathway. Alternately, cadmium may suppress placental HSD11B2 gene expression through activation of the estrogen receptor (ER). This contention is based on our previous findings that estrogen reduced 11{beta}-HSD2 expression and activity in cultured primary human trophoblast cells (73). Furthermore, cadmium activated ER in breast cancer cells (72) and mimicked the in vivo effects of estrogen in the rat uterus and mammary gland (31).

To determine whether other divalent metals had similar effects on placental 11{beta}-HSD2, we treated trophoblast cells with ZnCl2, MgCl2, and MnCl2. In contrast to cadmium, none of the three metals affected 11{beta}-HSD2 activity, suggesting that the decreased 11{beta}-HSD2 activity and expression were not general effects of heavy metals but were specific effects of cadmium. We also examined the effects of cadmium on the expression of HO-1, a known inducible target of this metal (51), to demonstrate that the decreased placental HSD11B2 gene expression was not due to cadmium-induced global downregulation of gene expression resulting from cytoxicity in trophoblast cells. As expected, cadmium evoked a concentration-dependent increase in the level of HO-1 mRNA. Furthermore, previous studies showed that cadmium, at 10 times higher concentration than that used in the present study, did not cause cytotoxic effects on cultured human trophoblast cells (38). Collectively, these findings suggest that cadmium exerts specific effects on placental 11{beta}-HSD2.

Our present study demonstrates that cadmium reduces placental 11{beta}-HSD2. If this occurs in the human placenta in vivo, it will lead to increased fetal exposure to maternal glucocorticoid and consequent FGR. It is noteworthy that previous studies have provided evidence for cadmium-induced potentiation of glucocorticoid receptor activation (20, 62). If cadmium exerts similar effects on the developing fetus, it would suggest that cadmium might not only result in increased glucocorticoid exposure due to reduced placental 11{beta}-HSD2 but also enhance the response to glucocorticoids, further exacerbating glucocorticoid-induced FGR. To determine whether other key constituents of tobacco smoke have similar effects on placental 11{beta}-HSD2, we treated trophoblast cells with nicotine and cotinine (a major metabolite of nicotine). Neither of the two compounds, at concentrations up to 1 mM, altered placental 11{beta}-HSD2 activity. These findings suggest that cadmium may be a key constituent of tobacco smoke that is involved in mediating the adverse effects of maternal smoking on fetal growth and development.

Human exposure to cadmium occurs primarily through dietary sources, cigarette smoking, and occupational exposure (24, 25). The average daily intake is estimated to be 10–60 µg/day (25, 52). The metal has a biological half-life of 10–30 years (52) and is known to accumulate in the placenta (29). In human placental tissues, the amount of cadmium was 12–535 and 6–270 ng/g in women from high [occupationally exposed or living in areas highly polluted by battery, smelters, or refineries (2, 5, 18)] and low (18, 19, 22, 23, 41) environmental exposures, respectively. Furthermore, mothers who smoked during pregnancy had significantly increased cadmium levels, approximately doubled that for nonsmoking mothers (9, 19, 37, 75). Thus the concentrations of cadmium found in human placenta in the general population (including both smokers and nonsmokers) are within those (0.25–1 µM = 45–180 ng/ml) that reduce placental 11{beta}-HSD2 activity and expression in the present study, supporting a role for the decreased placental 11{beta}-HSD2 in cadmium-induced FGR in the human.

Placental 11{beta}-HSD2 plays a key role in fetal development by protecting the fetus from exposure to high levels of maternal glucocorticoid. Consequently, molecules that downregulate placental 11{beta}-HSD2 can adversely affect fetal growth and contribute to FGR. The results of the present study suggest that cadmium is a candidate for such a role. At concentrations relevant to those in the human placenta in vivo, cadmium reduces placental 11{beta}-HSD2 activity and expression by suppressing HSD11B2 gene transcription. Thus our present study identifies placental 11{beta}-HSD2 as a novel molecular target of cadmium. It also reveals a molecular mechanism by which cadmium and smoking may exert their growth-restricting effects on the developing fetus.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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REFERENCES
 
This work was supported by the Canadian Institutes of Health Research.


   ACKNOWLEDGMENTS
 
We thank Dr. Daniel Hardy and Andrea Homan for their technical assistance and helpful discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Yang, Children's Health Research Institute, Rm. A5-132, Victoria Research Laboratories-Westminster Campus, 800 Commissioners Rd. East, London, ON, Canada N6A 4G5 (e-mail: kyang{at}uwo.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.


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 ABSTRACT
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 RESULTS
 DISCUSSION
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