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Am J Physiol Regul Integr Comp Physiol 281: R1966-R1974, 2001;
0363-6119/01 $5.00
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Vol. 281, Issue 6, R1966-R1974, December 2001

Inhibition of placental 11beta -hydroxysteroid dehydrogenase type 2 by catecholamines via alpha -adrenergic signaling

Sumita Sarkar1, Shu-Whei Tsai1, Tien T. Nguyen1, Michael Plevyak2,3, James F. Padbury1,3, and Lewis P. Rubin1,3

Departments of 1 Pediatrics and 2 Obstetrics and Gynecology and 3 Program in Fetal Medicine, Women & Infants Hospital of Rhode Island and Brown Medical School, Providence, Rhode Island 02905-2499


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The placenta expresses high levels of 11beta -hydroxysteroid dehydrogenase type 2 (11beta HSD2) that converts cortisol into inactive 11-keto metabolites and effectively protects the developing fetus from maternal cortisol during pregnancy. Impairment of this glucocorticoid barrier has adverse effects on fetal outcomes. A similar spectrum of adverse fetal effects is induced by antenatal stress during pregnancy. To examine the hypothesis that physiological stress may regulate placental 11beta HSD2 gene expression, we examined the effects of the catecholamines norepinephrine (NE) and epinephrine (E) on 11beta HSD2 expression in human trophoblastic cells. With the use of Northern blotting and semiquantitative RT-PCR, we determined that NE and E rapidly downregulate 11beta HSD2 steady-state mRNA levels in early- and late-gestation human trophoblasts and BeWo trophoblastic cells. Experiments using different adrenoceptor subtype-selective agonists and antagonists demonstrated that this catecholamine suppression of 11beta HSD2 mRNA expression is mediated via both alpha 1- and alpha 2-adrenoceptors and is independent of beta -adrenergic stimulation. To examine transcriptional regulation, BeWo cells were transiently transfected with a reporter construct in which an 11beta HSD2 human promoter sequence was inserted upstream of the luciferase gene. Treatment with 10-7 M NE decreased luciferase activity by ~60% (n = 3, P < 0.01). These results suggest the NE/E-mediated decrease in placental 11beta HSD2 gene expression is an instance of alpha -adrenoceptor-specific rapid transcriptional inhibition of an adrenergic target gene. This molecular mechanism may be involved in the deleterious effects of antenatal physiological stress on fetoplacental growth and development.

norepinephrine; epinephrine; gene transcription; trophoblast; pregnancy complications


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ANTENATAL MATERNAL STRESS is a risk factor for poor obstetric and infant outcomes including spontaneous abortion, congenital abnormalities, prematurity, and intrauterine growth restriction (IUGR) as well as susceptibility to cardiovascular and metabolic disease throughout life (4, 8, 10, 16, 22, 42). Although the mechanisms underlying these sequelae of antenatal stress are not well understood, in utero exposure to maternal glucocorticoids is a plausible mechanism for the influence of stress on fetal development.

Similar to physiological stress in the mother, excess fetal glucocorticoid exposure impairs growth, programs metabolic pathways, and predisposes to hypertension in adulthood (16). In human pregnancies, circulating levels of cortisol are severalfold higher in the mother than in the fetus (12) so that fetal development takes place in a relatively low-glucocorticoid milieu. In the rat, blocking maternal corticosterone secretion during restraint stress also blocks stress-induced effects in the offspring. Conversely, corticosterone administration to these mothers restores the adverse effects of stress (6).

During mammalian development, the maternal-fetal (transplacental) cortisol gradient is maintained by the enzyme 11beta -hydroxysteroid dehydrogenase (11beta HSD). The type 1 isoform, 11beta HSD1, acts mainly as an oxidoreductase to interconvert active glucocorticoids and their 11-dehydro metabolites (30). In mineralocorticoid target tissues, the type 2 isoform (11beta HSD2) unidirectionally converts cortisol and corticosterone to cortisone and 11-dehydrocorticosterone, respectively. 11beta HSD2 is the predominant isoenzyme in the placenta (10). Placental 11beta HSD2 immunoreactivity localizes to the maternal-facing syncytiotrophoblast (3, 9, 31, 46), consistent with a role as a barrier for fetal access to maternal glucocorticoids (37).

The similarities between the physiological consequences of fetal hypercortisolism and antenatal stress prompted us to test the hypothesis that placental 11beta HSD2 expression is regulated by the principal circulating stress-mediated catecholamines norepinephrine (NE) and epinephrine (E). Catecholamines bind multiple members of a family of alpha - and beta -adrenoceptors. These G protein-coupled seven-transmembrane receptors are categorized according to their ligand specificities, signal transduction heterogeneity, and molecular sequences. In the following study, we determined the effects of NE and E on trophoblast 11beta HSD2 expression and established which adrenoceptor subtype(s) mediate the influence of catecholamines on expression of this placental enzyme.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. L-Norepinephrine bitartrate (NE), L-epinephrine bitartrate (E), L-phenylephrine HCl (Phe), DL-propranolol, prazosin HCl (Pra), yohimbine HCl (Yoh), BHT 933 diHCl, ICI-118,551, pertussis toxin (PTX), and DMSO were purchased from Sigma/RBI (St. Louis, MO). The RT-PCR primers used in this study were purchased from Operon Technologies (Alameda, CA).

Primary culture of human term trophoblasts. Placental cytotrophoblasts (CTB) were isolated as previously described (43). Briefly, term human placentas were obtained after elective caesarean section. Portions of villous tissue were minced and digested two times with 0.125% trypsin (Sigma) and 0.02% deoxyribonuclease I (Sigma) in Hanks' balanced salt solution (HBSS) containing 0.8 mM MgSO4 and 25 mM HEPES (pH 7.4). CTB were isolated by centrifugation at 2,100 g for 20 min at 20°C through 5-70% Percoll (Amersham Pharmacia Biotech, Piscataway, NJ) step gradients. The CTB layer was collected, washed, and plated on fibronectin-coated dishes at a density of 10-15 × 106 cells/dish in DMEM (Life Technologies, Rockville, MD) containing 25 mM glucose and supplemented with 20% fetal bovine serum (FBS), 100 U/ml penicillin G, and 100 µg/ml streptomycin sulfate. Cells were cultured at 37°C in 95% air-5% CO2 before experimentation. The presence of nontrophoblastic cells was determined in methanol-fixed, acetone-permeabilized parallel cultures by immunocytochemistry with mouse monoclonal anti-vimentin antibody (Sigma) and assessment for fibroblast morphology. CTB purity in study cultures ranged from 90 to 97%. Trophoblasts differentiate morphologically in culture and show time-dependent accumulation of mRNAs for chorionic gonadotropin-alpha (alpha -hCG), beta -hCG, placental lactogen (hPL) and release of immunoreactive hCG, hPL, and progesterone (43). Trophoblast cultures were studied between 20 and 40 h.

Isolation and culture of first-trimester placental bed chorionic villi. Because specific effects of maternal stress and fetoplacental glucocorticoid exposure may have their origin early in postimplantation development (2, 10), we also examined the influence of catecholamine exposure on first-trimester placental villi. Placental tissues were collected from elective pregnancy terminations at 6-9 wk of gestation in accordance with procedures approved by the Institutional Review Board of Women & Infants Hospital. Tissue was rinsed in sterile HBSS, and chorionic villi with adherent maternal decidua were separated, minced in serumless DMEM supplemented with antibiotics, and maintained overnight at 37°C in 95% air-5% CO2 before experimentation.

Culture of BeWo (b30 clone) cells. BeWo human choriocarcinoma cells and the b30 subline exhibit phenotypic characteristics similar to first-trimester invasive trophoblasts (28, 43). BeWo cells (ATCC, Manassas, VA) and the BeWo b30 clonal subline (originally provided by Dr. Alan L. Schwartz, Washington Univ.) were maintained in DMEM-10% FBS and used for assay of catecholamine-regulated 11beta HSD2 mRNA expression or transient transfections.

RNA extraction and Northern blot analysis. Total cellular RNA was isolated from placental trophoblast cells treated with specific test ligands using the one-step method (13) with modifications (44). RNA samples (20 µg/lane) were electrophoresed in 1.4% agarose gels containing 2.2 M formaldehyde and then transferred to nylon membranes (GeneScreen; Dupont-NEN, Boston, MA) by capillary blotting. Northern blot hybridization was carried out using a highly homologous rat 11beta HSD2 cDNA [gift from Dr. Celso Gomez-Sanchez, University of Missouri (Ref. 53)] subcloned into pcDNA3 (Invitrogen, Carlsbad, CA) and linearized to prepare antisense cRNA probes by in vitro transcription (Promega, Madison, WI). All blots were stripped and rehybridized with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) riboprobe (44) to normalize for loading and transfer discrepancies.

Densitometric analysis of autoradiograms was performed on a high-resolution laser beam densitometer, and the scanned images were analyzed using National Institutes of Health (NIH) Image v1.9. In all densitometric measurements, the amount of RNA electrophoresed was within a range in which the intensities of the bands were found to increase linearly with the amount of RNA.

RT-PCR. In instances where 11beta HSD2 mRNA expression was low (BeWo cells) and for certain primary trophoblast experiments, we used semiquantitative RT-PCR (33). The latter approach was optimized for linearity of amplification in preliminary experiments for human 11beta HSD2 detection. Briefly, total RNA was isolated as described above, and RNA integrity was checked by agarose gel electrophoresis in the presence of 2.2 M formaldehyde. For first-strand cDNA synthesis, 1 µg of total RNA was treated with RNase-free DNase I (Stratagene, San Diego, CA) and diluted in a reaction mixture (20 µl) containing 20 mM Tris · HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 U Superscript II RT (Life Technologies), 10 mM dithiothreitol, 2-deoxynucleotide 5'-triphosphates (dNTPs; 200 µM each of dATP, dCTP, deoxyguanosine 5'-triphosphate, and thymosine triphosphate), and 0.5 µg standard oligo-dT primers and was reverse transcribed at 42°C for 1 h. For each RT, a blank was performed consisting of all the reagents with water substituted for RNA. DNA amplification was carried out using 2 µl of the RT products in a 50-µl reaction volume containing 10 mM Tris · HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 1.25 U cloned Thermus aquaticus DNA polymerase (Amplitaq, Perkin-Elmer/Cetus, Norwalk, CT), 200 mM of each dNTP, and 40 pmol of either the 11beta HSD2, 11beta HSD1, or GAPDH primer sets. PCR was performed in a programmable thermal cycler (MJ Research, Watertown, MA).

For 11beta HSD2 PCR, sequence-specific oligonucleotide primers (forward primer, 5'-TGCTGCAGATGGACCTGACCAA-3'; reverse primer, 5'-TAGTAGTGGATGAAGTACATGAGC-3') were amplified at 90°C, 30 s; 55°C, 30 s; 72°C, 60 s; 28 cycles; followed by final extension, 72°C, 10 min. For 11beta HSD1, the DNA primers were 5'-GCTCTGCCTGGGTTACTACTA-3' (forward) and 5'-CAGAAGTGGAGTCCAAGATGAT-3' (reverse). The 11beta HSD1 amplification program consisted of 90°C, 50 s; 60°C, 50 s; 72°C, 60 s; 35 cycles; followed by final extension, 72°C, 10 min. Coamplification with GAPDH primers was included to control for efficiency of RNA isolation and cDNA synthesis. To confirm identity, PCR products were gel purified, subcloned, and sequenced by the dideoxy chain termination procedure. One-fifth of the PCR solution was added to 3 µl of gel-loading buffer (40% sucrose, 0.05% bromophenol blue, 0.5% SDS, 100 µM EDTA), electrophoresed in 1.8% agarose gels containing 0.5 µg/ml ethidium bromide, and photographed. Scanned images were quantified using Photoshop v 4.5 (Adobe Systems, San Jose, CA) and NIH Image v1.9 software.

For RT-PCR detection of adrenoceptor subtype expression in placenta and trophoblast cells, we used previously described type-specific primer sets for human beta 1- and beta 2-adrenoceptors (17), predicting 524- and 372-bp PCR products, respectively. The beta 1 amplification program consisted of 94°C, 30 s; 58°C, 30 s; 72°C, 60 s; 30 cycles. The beta 2 amplification program consisted of 94°C, 30 s; 50°C, 30 s; 72°C, 60 s; 29 cycles. For alpha 1-adrenoceptor subtype detection, human alpha 1A-, alpha 1B-, and alpha 1D-primer sets (50) were used to generate amplicons of 501, 530, and 354 bp, respectively. PCR programs consisted of 94°C, 60 s; 50°C (55°C for alpha 1B), 30 s; 72°C, 60 s; 30 cycles. For detection of alpha 2-adrenoceptor subtypes, human alpha 2A-, alpha 2B-, and alpha 2C-primers (39) were used to generate amplicons of 297, 391, and 574 bp, respectively. The alpha 2-receptor PCR programs consisted of 95°C, 45 s; 65°C, 45 s; 72°C, 60 s; 30 cycles.

Transient transfection and gene expression assays. A 1,788-bp fragment of the 5'-flanking region of the human 11beta HSD2 gene subcloned into the pGL2 (Promega) luciferase reporter vector [provided by Dr. Anil Agarwal, UT Southwestern Medical School (Ref. 1)] was transiently transfected into BeWo clone b30 cells using Lipofectin (Life Technologies). Briefly, 0.5 × 106 cells/well were cultured in 24-well plates in the presence of DMEM-10% FBS. After being washed, cells in DMEM-10% FBS were transiently cotransfected in quadriplicate with 0.5 µg of 11beta HSD2-luciferase plasmid DNA and 1 µg of a cytomegalovirus promoter (pCMV) beta -galactosidase plasmid (Promega) to monitor transfection efficiency. Control wells were transiently transfected with a promoterless pGL2 vector. After 24 h, cells were washed with PBS and treated with serumless DMEM and the various experimental agents. At the end of each incubation, cells were again washed with PBS, lysed in 150 µl of lysis buffer (BD PharMingen, San Diego, CA), and scraped from the dishes. After centrifugation at 13,000 g at 4°C for 5 min, luciferase and beta -galactosidase activities were determined according to the manufacturers' protocols. Luciferase activity was measured using a Monolight 3010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). Values were corrected for beta -galactosidase activity and normalized to protein concentration. Transfection efficiencies exceeded 60%.

Data analysis. Data are reported as means ± SE. Significance tests (Student's unpaired t-test or ANOVA) were performed to compare values of samples treated with the different experimental conditions. P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of NE on 11beta HSD2 mRNA expression. We used semiconfluent trophoblast cultures to determine whether NE has time- and concentration-dependent effects on placental 11beta HSD2 mRNA levels. Trophoblasts were treated with NE (10-7 M) or vehicle (10-4 M ascorbic acid) for 0.5, 1, 2, and 24 h, and RNA was assayed for 11beta HSD2 expression. Figure 1A indicates that NE inhibited 11beta HSD2 mRNA levels at 30 min, the earliest time point examined (lanes 1 and 2). After 1-h exposure to NE, 11beta HSD2 mRNA levels decreased further to ~50% of control (Fig. 1A, lanes 3 and 4, and 1B). With longer NE treatment, placental 11beta HSD2 mRNA levels returned to baseline. In further experiments, we verified that the inhibitory effects of NE and E on trophoblast 11beta HSD2 mRNA accumulation are concentration dependent. Threshold effects were detected at 10-9 M and maximal effects at 10-7-10-6 M (not shown).


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  		Fig. 1.   Effect of norepinephrine (NE) on the expression of 11beta -hydroxysteroid dehydrogenase type 2 (11beta HSD2) mRNA. Human term placental trophoblasts were isolated as described in MATERIALS AND METHODS and maintained in DMEM-20% fetal bovine serum (FBS) overnight. Before experimentation, cells were washed and maintained in serumless medium for 2 h followed by treatment with vehicle (C) or NE (10-7 M) in 10-4 M ascorbic acid for 0.5, 1, 2, and 24 h. At each time point, monolayers were lysed for RNA extraction. A, top: twenty micrograms per lane of total RNA were electrophoresed and blotted. This Northern blot was hybridized with an [alpha -32P]UTP-labeled antisense cRNA probe for 11beta HSD2. In this representative blot, NE induced a time-dependent decrease in trophoblast 11beta HSD2 mRNA accumulation. A, middle: reprobing of this stripped blot with chorionic gonadotropin-alpha (alpha hCG) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cRNA probes, respectively. In contrast to the effect on 11beta HSD2, NE caused a slower, time-dependent accumulation of alpha hCG mRNA. A, bottom: GAPDH hybridization and ethidium bromide ribosomal RNA fluorescence photomicrograph indicate uniform RNA sample loading and transfer. B: densitometric Northern blot quantitation of 11beta HSD2 mRNA accumulation was measured in trophoblast cultures treated with C or NE for 1 h (n = 4 different placental isolations; **P < 0.01). Values are means ± SE.

We next established that this decrease in 11beta HSD2 mRNA levels was not due to a generalized effect on trophoblast gene expression. The Northern blots were stripped and reprobed for expression of alpha -hCG. This placental gene is positively transcriptionally regulated by catecholamines via a cAMP-dependent pathway (43). As shown in Fig. 1A, NE induced a time-dependent increase in alpha -hCG mRNA accumulation. The NE-stimulated increase in alpha -hCG expression was also delayed, compared with the rapid NE-stimulated decline in trophoblast 11beta HSD2 mRNA levels.

Effect of NE on 11beta HSD2 mRNA accumulation in first-trimester chorionic villi. In the first weeks of pregnancy, trophoblasts from the blastocyst invade the endometrium and myometrium, open and remodel the uterine vessels, and become exposed to high levels of maternal glucocorticoids. Consequently, we also examined the influence of NE on 11beta HSD2 expression in early-gestation trophoblast. Chorionic villus explants containing predominantly invasive trophoblast and adherent maternal decidua (6-9 wk) were incubated with NE (10-7 M) as described for primary trophoblast cell cultures. Figure 2 indicates NE treatment resulted in an ~50% decrease in 11beta HSD2 mRNA expression by 1 h. These results, obtained by semiquantitative RT-PCR, are similar to the inhibition of 11beta HSD2 mRNA levels determined by Northern analysis (Fig. 1) and RT-PCR in term trophoblast.


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  		Fig. 2.   NE inhibits 11beta HSD2 mRNA accumulation in early-gestation trophoblast. The RT-PCR fluorescence photomicrographs indicate the effects in 1 representative experiment. Placental villus explants comprised principally of trophoblast with adherent decidua were treated with C or 10-7 M NE for 1 h. RNA was isolated and subjected to RT-PCR as described in MATERIALS AND METHODS using specific primers for 11beta HSD2 (top), 11beta HSD1 (bottom), and the constitutively expressed GAPDH. The DNA amplicons were electrophoresed in 1% agarose TAE gels stained with EtBr and photographed. PCR conditions were adjusted to permit quantitation of amplified signals. The DNA size markers in each image (right) indicate 100-bp intervals. The bright central band indicates 500 bp.

During early gestation, placental trophoblast expresses the 11beta HSD2 isoform and adjacent decidua expresses the 11beta HSD1 glucocorticoid oxidoreductase nearly exclusively (2). Because local cortisol levels are regulated by the balance between the two placental bed 11beta HSD isoforms, we simultaneously determined the effects of NE on 11beta HSD1 expression in the placental explants using RT-PCR. Under conditions where NE significantly downregulated 11beta HSD2 expression, there was little effect on 11beta HSD1 mRNA levels (Fig. 2). In agreement with previous observations (2), when the two tissue types were separately maintained, we detected only 11beta HSD2 PCR product in trophoblastic villi and only 11beta HSD1 in decidua.

Catecholamines downregulate trophoblast 11beta HSD2 gene expression via alpha -adrenoceptors. Adrenoceptors (alpha  and beta ) are divided into functional subtypes based on rank order of potency and specificity of agonists and antagonists coupling to different signal transduction pathways and, more recently, molecular cloning studies (11, 14). To determine whether the effects of NE and E on placental 11beta HSD2 gene expression are receptor dependent and, if so, via which adrenoceptor subtype(s), trophoblasts were incubated with NE, E, or beta -, alpha 1-, or alpha 2-adrenoceptor-selective agonists. The adrenergic agonists were used in concentrations of 10-7 M to ensure receptor selectivity (26).

Incubation either with an alpha 1-agonist (Phe) or an alpha 2-agonist (BHT 933) for 1 h partially mimicked the inhibitory effect of NE on trophoblast 11beta HSD2 mRNA (Fig. 3). Brief treatment (1 h) with the beta -agonist isoproterenol (Iso) had no effect on 11beta HSD2 mRNA accumulation (Fig. 3). In contrast, longer incubations with forskolin (>4 h) increased 11beta HSD2 expression three- to fourfold, consistent with previous observations of cAMP-dependent 11beta HSD2 stimulation (40, 47).


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  		Fig. 3.   Effects of specific adrenergic agonists on trophoblast 11beta HSD2 mRNA expression. Term trophoblast cultures were incubated with C, NE, isoproterenol (Iso), phenylephrine (Phe), or BHT 933 (BHT) for 1 h. All agonists were 10-7 M final concentration. Inset: representative Northern blot hybridized with an 11beta HSD2 [alpha -32P]UTP-labeled antisense cRNA probe. The histogram depicts densitometric analysis of the 11beta HSD2 mRNA abundance normalized to cohybridized GAPDH mRNA expression for 3 different experiments (means ± SE). *P < 0.05 compared with control; **P < 0.01 compared with control.

These findings suggest the rapid catecholamine-mediated suppression of placental 11beta HSD2 mRNA levels is mediated by signaling via trophoblast alpha 1- and alpha 2-adrenoceptors. Because the trophoblast-specific expression of different placental adrenoceptors has not been well characterized, we next determined whether alpha 1- and alpha 2-receptors are expressed in term placental tissue (PT) and purified trophoblasts using RT-PCR and subtype-specific oligomer primer sets.

Three human alpha 1-adrenoceptor subtypes, alpha 1A, alpha 1B, and alpha 1D, have been identified by molecular cloning techniques (11). In isolated trophoblasts (CTB), we detected only alpha 1B expression (Fig. 4, top). All three alpha -adrenoceptor isoforms were detected in PT, presumably owing to the presence of endothelial and smooth muscle cells. We also detected trophoblast expression of alpha 2A- and alpha 2B- but not alpha 2C-receptors (Fig. 4, middle). In addition, we verified that trophoblast expresses the beta 1- and beta 2-adrenoceptors (Fig. 4, bottom).


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  		Fig. 4.   Detection of alpha 1-, alpha 2-, and beta -adrenoceptor subtypes by RT-PCR in human trophoblast cultures on day 2 [cytotrophoblasts (CTB) and placental tissue (PT)]. Two micrograms of RNA were subjected to RT-PCR as described in MATERIALS AND METHODS using specific primers for alpha 1-adrenoceptor subtypes (top), alpha 2-adrenoceptor subtypes (middle), and beta -adrenoceptor subtypes (bottom). The DNA amplicons (one-fifth of each PCR reaction solution volume) were electrophoresed in 1% agarose TAE gels stained with EtBr and photographed. Trophoblast specifically expresses the alpha 1B-, alpha 2A-, alpha 2B-adrenoceptor subtypes and beta -adrenoceptors.

We then further investigated the requirement for alpha 1- and alpha 2-adrenergic signaling by a combinatorial approach with subtype-selective receptor antagonists. All antagonists were used in concentrations (10-6 M) 10-fold in excess to the natural ligands (NE, E, 10-7 M). The beta 2-isoform is the principal placental beta -adrenoceptor (45). Trophoblast cultures were coincubated with the beta 2-specific adrenergic antagonist ICI-118,551 plus NE to assess the alpha -adrenergic effect on 11beta HSD2 gene expression. Figure 5 indicates that ICI-118,551 alone did not alter 11beta HSD2 mRNA accumulation. However, in the presence of beta 2-blockade with ICI-118,551, NE treatment resulted in rapid suppression of steady-state 11beta HSD2 mRNA levels. The magnitude of this suppression was significantly greater than the effect of NE in the absence of beta -blockade (Fig. 5), supporting a signaling model in which regulation of 11beta HSD2 mRNA expression by alpha - and beta -adrenoceptors is opposed. Similar results were obtained using the nonselective beta -antagonist propranolol (not shown).


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  		Fig. 5.   Effect of beta -adrenergic blockade and pertussis toxin (PTX) on NE-mediated trophoblast 11beta HSD2 mRNA expression. Trophoblast cultures were incubated for 1 h with C, 10-7 M NE, 10-6 M beta 2-adrenergic antagonist ICI-118,551 (ICI), ICI + NE, 100 ng/ml PTX, or PTX + NE. Cells were pretreated for 2 h with PTX followed by the test reagents. RNA samples were isolated, electrophoresed, blotted, and hybridized with an 11beta HSD2 [alpha -32P]UTP-labeled antisense cRNA probe. The histogram depicts densitometric analysis of the 11beta HSD2 mRNA abundance normalized to cohybridized GAPDH mRNA expression for 3 different experiments (means ± SE). **P < 0.01 compared with control; ***P < 0.001 compared with control.

alpha 1-Adrenoceptors interact with Gq/p family Galpha subunits leading to activation of phospholipase C, hydrolysis of phosphoinositides, and mobilization of cytosolic Ca2+. alpha 2-Adrenoceptors, in contrast, couple to Gi/o family Galpha subunits with the principal effect of inhibiting adenylyl cyclase and are sensitive to PTX inactivation. Therefore, we used PTX blockade of alpha 2-adrenoceptor-coupled Gi to determine the contribution of alpha 1-adrenoceptors. When isolated trophoblasts were incubated with PTX (100 ng/ml) alone, no effect on 11beta HSD2 mRNA accumulation was detected (Fig. 5). However, when trophoblasts were preincubated with PTX followed by exposure to NE, the NE-induced decrease in expression of 11beta HSD2 mRNA was partially blocked (Fig. 5). This result is consistent with our findings that the alpha 1- and alpha 2-selective agonists each induced decreases in trophoblast 11beta HSD2 mRNA levels.

To distinguish further between alpha 1- and alpha 2-adrenoceptor-dependent effects, trophoblasts were subjected to beta -blockade using propranolol plus blockade either of alpha 1-adrenoceptors with Pra or alpha 2-adrenoceptors with Yoh, followed by incubation with NE or E. In the presence of either beta  + alpha 1- or beta  + alpha 2-adrenoceptor blockade, NE and E still decreased trophoblast 11beta HSD2 mRNA levels. It is notable that these decreases in 11beta HSD2 mRNA were smaller than those seen after treatment with NE or E only or treatment with the nonselective alpha -adrenoceptor agonist Phe. Taken together, these findings support a model for cross talk or convergence between alpha 1- and alpha 2-adrenoceptor signaling pathways leading to suppressed 11beta HSD2 gene expression.

BeWo choriocarcinoma cells show catecholamine-mediated 11beta HSD2 mRNA inhibition. We also investigated the parent BeWo and the BeWo b30 clonal subline for NE-mediated suppression of 11beta HSD2 mRNA levels. Similar to the results for early- and late-gestation normal trophoblast, BeWo and BeWo b30 cells showed time-dependent, rapid decreases in 11beta HSD2 expression in response to NE (Fig. 6). No 11beta HSD1 expression in the BeWo lines was detected by RT-PCR (not shown). Because this cell line retains catecholamine-regulated 11beta HSD2 expression, BeWo b30 cells were used for transient transfection experiments described below.


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  		Fig. 6.   NE inhibits 11beta HSD2 mRNA accumulation in BeWo trophoblastic cells. BeWo cell monolayers were incubated with C or 10-7 M NE for the indicated times (0.5 to 24 h). RNA was isolated and subjected to RT-PCR as described in MATERIALS AND METHODS using specific primers for 11beta HSD2 and the constitutively expressed GAPDH. The DNA amplicons were electrophoresed in 1% agarose TAE gels stained with EtBr and photographed. PCR conditions were adjusted to permit quantitation of amplified signals. The histogram depicts the normalized 11beta HSD2 expression from 1 of 3 similar experiments. The time course of NE-inhibited 11beta HSD2 mRNA expression is similar to that in a normal trophoblast.

Downregulation of trophoblast 11beta HSD2 mRNA by NE is transcriptionally mediated. To confirm whether the rapid decline in steady-state 11beta HSD2 mRNA represents transcriptional repression, BeWo b30 cells were transiently transfected with a proximal promoter sequence of the human 11beta HSD2 gene (-1,788 luciferase) linked to the luciferase gene in a pGL2 reporter gene construct. After transfection, cells were treated with NE for 1 h. Figure 7 demonstrates that NE significantly decreased expression of the reporter construct (measured as normalized luciferase activity) by ~60%.


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  		Fig. 7.   Transient transfection of an 11beta HSD2 proximal promoter luciferase reporter plasmid into BeWo cells. BeWo cells maintained in 24-well plates were cotransfected with the 11beta HSD2 proximal promoter luciferase reporter and beta -galactosidase cDNAs as described in MATERIALS AND METHODS. After transfection, wells were treated with C or 10-7 M NE for 1 h. Cells were lysed and assayed for luciferase and beta -galactosidase activities to correct for differences in transfection efficiency. The histogram depicts the corrected luciferase activity for 3 independent experiments, each performed in triplicate, **P < 0.01.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this report, we show that catecholamines in a concentration range similar to the circulating NE and E concentrations during pregnancy (27) downregulate 11beta HSD2 gene expression in human placental trophoblast. Trophoblast is exposed to NE and E throughout pregnancy. Phenylethanolamine N-methyltransferase, which catalyzes NE to E and is expressed principally in the adrenal gland, is also active in uterine and fetal membrane tissues (24, 35). Our findings suggest the presence of a molecular link between elevated catecholamine levels and the regulation of fetoplacental glucocorticoid metabolism.

The multiple target cell responses to NE and E are mediated by distinct adrenoceptor subtypes coupled to different signal transduction pathways (11). beta -Adrenergic stimulation characteristically induces adenylyl cyclase-dependent cAMP production (20). The adenylyl cyclase activator forskolin stimulated 11beta HSD2 mRNA accumulation, but the onset of this effect was delayed compared with the alpha -adrenergic-mediated rapid decrease in 11beta HSD2 mRNA levels. In fact, coupling of different beta - and alpha -adrenoceptor types to stimulatory (Gs) and inhibitory (Gi) Galpha subunits dually regulates adenylyl cyclase activity. Both signaling pathways are functional in the placenta (20), and this organ is a rich source of both alpha - and beta -adrenoceptors (19, 38, 45), making analysis of trophoblast responses to catecholamine stimulation complex.

In tissues or cells expressing multiple adrenoceptor subtypes, such as the placenta and trophoblast, it may be difficult to distinguish unequivocally among adrenoceptor subtype-specific effects. Pharmacological studies have suggested alpha 2-adrenoceptors are the principal alpha -adrenoceptors in human PT (5). Similarly, functional alpha 2A- and alpha 2B-receptors have been identified by competition binding analysis in membrane fractions isolated from human PT (18). However, the expression and heterogeneity of alpha -adrenoceptors specifically in the trophoblast have not been explored. Therefore, we assayed cultured trophoblasts and trophoblast explants for subtype-specific receptor expression by RT-PCR. We detected trophoblast-specific expression of alpha 1B-, alpha 2A-, and alpha 2B-adrenoceptors. Because our functional data indicate that both alpha 1- and alpha 2-receptors participate in catecholamine-mediated inhibition of 11beta HSD2 mRNA levels, presumably these effects are transduced by the trophoblast alpha 1B-adrenoceptor and one or both trophoblast alpha 2-adrenoceptor subtypes.

Of interest, binding data (5, 18) reveal that concentrations of placental alpha 2-adrenoceptors decline with advancing gestational age so that there is a general shift in the placental alpha /beta -adrenoceptor ratio toward increasing beta -receptor dominance. In the course of our experiments, we found that NE or E consistently suppressed 11beta HSD2 mRNA levels in first-trimester trophoblast cultures, but the extent and duration of the catecholamine effect were more variable in term trophoblast, possibly reflecting a greater beta -receptor effect. Similarly, although direct activation of adenylyl cyclase by forskolin consistently stimulated an increase in 11beta HSD2 mRNA accumulation by 4-24 h, treatment of term trophoblast cultures with the beta -adrenergic agonist Iso increased 11beta HSD2 mRNA levels only in some trophoblast preparations.

We have not tested the relationship between adrenoceptor subtype density and the magnitude and duration of catecholamine suppression of trophoblast 11beta HSD2 mRNA levels. However, it is relevant that the shift toward beta -adrenoceptor dominance as pregnancy advances does parallel increases in maternal circulating levels of glucocorticoid and placental 11beta HSD2 activity (41). It is possible, therefore, that alpha -adrenergic-mediated 11beta HSD2 suppression might be particularly important for the stress-mediated effects of glucocorticoids on the developing uteroplacental bed and on permeability of the placental glucocorticoid barrier during critical periods for fetal development.

In recombinant systems, alpha 2-adrenoceptors preferentially transduce signals through inhibitory, PTX-sensitive Gi/Go proteins. This leads to a decrease in cAMP that can inhibit voltage-gated Ca2+ channels and open K+ channels (14). We found that preincubation with PTX attenuated, but did not completely abolish, catecholamine-induced inhibition of 11beta HSD2 mRNA. This observation supports a contribution of both alpha 1- and alpha 2-receptor signaling in suppressing trophoblast 11beta HSD2 mRNA levels. We also performed transient transfections of an 11beta HSD2 promoter-luciferase gene fusion construct in trophoblastic cells. We found that catecholamine treatment resulted in a rapid transcriptional inhibition of the fusion construct.

In primary trophoblasts and BeWo cells, NE and E produced an ~50% decline in the expression of 11beta HSD2 mRNA within 1 h of treatment. This downregulation of trophoblast 11beta HSD2 mRNA levels in response to continuous ligand exposure reversed after several hours, consistent with ligand-dependent desensitization. Short-term exposure (several minutes) to beta 2-agonists leads to receptor phosphorylation by both cAMP-dependent protein kinase A and specific beta 2-adrenoceptor kinase, and a more prolonged exposure to an agonist leads to changes in receptor transcription or mRNA stability (8). The human alpha 2-adrenoceptor subtypes undergo similar patterns of agonist-dependent desensitization during short- and long-term continuous exposure to agonists (15, 50).

Perspectives

Despite the importance of trophoblast 11beta HSD2 during development, relatively few regulators of this placental enzyme have been identified. Retinoic acid (49), forskolin, and dibutyryl cAMP (40, 47) stimulate 11beta HSD2 activity and/or mRNA expression, whereas progesterone, estrogen (47), and nitric oxide (48) inhibit placental 11beta HSD2. To our knowledge, no previous studies have explored the effects of catecholamines on 11beta HSD2 expression in placental cells. We have determined that the stress-mediated catecholamines NE and E induce rapid, reversible decreases in 11beta HSD2 mRNA expression.

Interestingly, brief (45 min) sessions of restraint stress in pregnant rats also cause rapid increases in maternal and fetal corticosterone levels obtained during (51) and immediately following (52) a stress session, but corticosterone levels normalize in samples obtained from fetuses 30-165 min after the end of a stress session (51, 52). The rapid onset and short duration of the fetal corticosterone surge are similar to our in vitro observations of the catecholamine-induced decline and return to baseline of placental 11beta HSD2 gene expression.

The hypothesis that excess exposure of the fetoplacental unit to maternal glucocorticoids reduces birth weight and programs subsequent metabolic and neurodevelopmental development has been supported by studies using pharmacological blockade of 11beta HSD2 activity (34) and observations in human hypertension caused by 11beta HSD2 mutations (29, 36). The finding of attenuated placental 11beta HSD2 activity in IUGR also suggests that excess glucocorticoid exposure may contribute to impaired fetal growth (46). Therefore, placental 11beta HSD2 activity may be a common pathway by which environmental factors, such as maternal stress, alter fetoplacental development and program cardiovascular and metabolic disorders in later life, including hypertension (32).

Physiological stresses induce activation of the sympathoadrenal system and the hypothalamo-pituitary-adrenal axis. Although we did not measure cortisol metabolism in this study, our data support a model in which prenatal stress leading to elevated maternal and/or fetal catecholamine levels may decrease the placental glucocorticoid barrier and, thereby, increase fetal exposure to maternal glucocorticoids. These findings suggest a molecular mechanism underlying the connection between elevated catecholamine and fetal cortisol levels during physiological stress. This mechanism may, in part, explain how surges in circulating catecholamine levels might impair implantation, placental bed formation, and fetal growth. Future studies will address this issue.

Other instances of catecholamine-inhibited gene transcription [e.g., plasminogen activator inhibitor-1 gene in human adipose tissue (21) and macrophage inflammatory protein-1alpha (23)] involve beta -adrenergic, cAMP-dependent pathways. Demonstration of an alpha -adrenergic-dependent, rapid transcriptional inhibition is novel. The specific pathway(s) involved may have broader biological importance.


    ACKNOWLEDGEMENTS

The authors thank Drs. A. Agarwal and P. White for supplying the human 11beta HSD2 reporter construct, Dr. C. Gomez-Sanchez for the 11beta HSD2 cDNA, Dr. A. L. Schwartz for the BeWo b30 cell line, V. Hovanesian for image analysis, and R. Allen for assistance with manuscript preparation. In addition, we thank Drs. A. Brem, M. Malee, and D. Morris for helpful discussions.


    FOOTNOTES

This work was supported by National Institutes of Health Grant HD-11343.

Address for reprint requests and other correspondence: L. P. Rubin, Dept. of Pediatrics, Women and Infants Hospital, 101 Dudley St., Providence, RI 02905-2499 (E-mail: Lewis_Rubin{at}brown.edu).

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 12 May 2001; accepted in final form 23 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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