Expression and Regulation of Glucocorticoid Receptor in Human Placental Villous Fibroblasts
http://www.100md.com
《内分泌学杂志》
Departments of Obstetrics and Gynecology (M.-J.L., S.S.K.), Microbiology (Z.W., M.J.G.), Pathology (H.Y.), Urology (N.S., S.K.L.), and Pharmacology (S.K.L.), New York University School of Medicine, New York, New York 10016
Department of Obstetrics and Gynecology (Y.M., L.Y., S.G.), Yale University School of Medicine, New Haven, Connecticut 06520-8063
Department of Pathology (R.N.B.), Weill Medical College of Cornell University, New York, New York 10021
Abstract
The human placenta is a glucocorticoid (GC)-responsive organ consisting of multiple cell types including smooth muscle cells, fibroblasts, and trophoblast that demonstrate changes in gene expression after hormone treatment. However, little is known about the relative expression or activity of the GC receptor (GR) among the various placental cell types. Normal term human placentas were examined by immunohistochemistry using either GR phosphorylation site-specific antibodies that are markers for various activation states of the GR or a GR antibody that recognizes the receptor independent of its phosphorylation state (total GR). We found strong total GR and phospho-GR immunoreactivity in stromal fibroblasts of terminal villi, as well as perivascular fibroblasts and vascular smooth muscle cells of the stem villi. Lower levels of both total GR and phospho-GR were found within cytotrophoblast cells relative to fibroblasts, whereas syncytiotrophoblast showed very little total GR or phospho-GR immunoreactivity. This pattern holds true for immunoblot analysis of extracts from cell fractions cultured ex vivo. In cultured placental fibroblasts, phosphorylation of GR increased upon short-term GC treatment, consistent with a role for GR phosphorylation in receptor transactivation. Total GR levels were reduced by nearly 90% after long-term hormone treatment; however, this down-regulation was independent of changes in GR mRNA levels. These findings demonstrate that GR levels in fibroblasts can be modulated by changes in hormone exposure. Such cell type-specific differences in GR protein expression and phosphorylation may provide the means of differentially regulating the GC response among the cells of the human placenta.
Introduction
PREVIOUS STUDIES BY our laboratory and others (1, 2, 3) have documented that glucocorticoid (GC) is a key regulator of placental gene expression. GC-regulated genes in placenta include human chorionic gonadotropin (hCG) (4), CRH (5), plasminogen activator inhibitor-1 (6), fibronectin (7), and glucose transporters (8), demonstrating that the human placenta is a GC-responsive organ. Of particular relevance to the current study are the findings that GCs enhance the expression of extracellular matrix (ECM) proteins by placental fibroblasts (FIB) (i.e. stromal cells from the villous core of the placenta) in human and nonhuman primates (3, 7). The ECM synthesized by these placental cells plays an important role in maintaining tissue structure by providing a biological support for cytotrophoblast (CT) and syncytiotrophoblast (SCT) layers on the surface of the villus and fetal capillaries below (9, 10, 11, 12). FIB are present in both large stem villi, which form the main trunk of the villous tree (300 μm in diameter), and terminal villi, which are the final villous branches (50 μm in diameter) that are the main site of nutrient exchange between mother and fetus (13). Excessive synthesis of ECM proteins by placental FIB has been noted in pregnancies complicated by intrauterine growth restriction (IUGR) (14, 15). These pregnancies are also characterized by aberrantly high periplacental levels of GC (16, 17, 18), but the significance of this observation in the pathophysiology of IUGR remains to be elucidated.
Although several studies have examined GC-mediated gene regulation in human placenta (2, 3, 4, 5), little is known concerning the expression and regulation of placental GC receptor (GR). It has been demonstrated that human placenta expresses GR (19, 20, 21), but patterns of cell type-specific expression and regulation have not been established. GR is expressed in virtually every mammalian cell but controls distinct subsets of responsive genes, depending on cell type (22). The human GR (hGR) has two isoforms, GR and GR, that arise due to alternative splicing of the 3' terminus of the GR gene (23). The GR isoform was reported to be transcriptionally inactive and inhibited GR-mediated effects (24).
Besides being a ligand-dependent transcription factor, GR is also subject to posttranslational modification through phosphorylation (25). GR is phosphorylated in the absence of hormone, and additional phosphorylation events occur after agonist binding. Three major serine sites are phosphorylated within the N-terminal region of the receptor that are involved in transcriptional regulation (S203, S211, and S226 using the human numbering scheme). We have also identified the kinases that target these sites in vitro: cyclin E/Cdk2 and cyclin A/Cdk2 phosphorylate S203 and S211, respectively, whereas c-Jun N-terminal kinases phosphorylate S226 (26). GR phosphorylation may determine target gene activity, cofactor interaction, strength and duration of receptor signaling, or receptor stability. For example, phosphorylation at S211 has been associated with nuclear translocation and transcriptional activation of GR after hormone treatment (27). There is some evidence to suggest that phosphorylation at S203 is a determinant of ligand-dependent down-regulation of GR (27), but the exact mechanisms have not been clearly defined. These findings strongly suggest that changes in GR phosphorylation render the receptor differentially responsive to GC treatment. The phosphorylated isoforms of GR can be considered surrogates for various transcriptionally active states of the receptor (25). We have recently generated novel GR phosphorylation-specific antibodies to these phospho-serine sites to allow a detailed analysis by immunohistochemistry of GR phospho-isoform expression in vivo without the use of radioactivity (27).
In addition to the rapid (1–2-h) effects of GC on GR phosphorylation, it has been demonstrated that long-term (>4-h) treatment with GC promotes down-regulation of GR in several cell types (28, 29, 30). GC-dependent suppression of GR (i.e. homologous down-regulation) has been demonstrated to be a mechanism to limit hormone responsiveness (31, 32). Although not completely elucidated, the cellular mechanisms of homologous down-regulation include inhibition of GR transcription (15, 16) and decreased GR protein stability (21, 33, 34). These pathways also appear to be cell-type specific (32).
Therefore, the purpose of the present study was to establish the patterns of expression of total GR and phospho-GR in human placental tissue and then to examine their regulation in placental FIB in vitro.
Materials and Methods
Tissue procurement
Placental tissue used for immunohistochemical and cell-culture studies was obtained from uncomplicated pregnancies from normal term (36–41 wk gestation) with appropriately grown, singleton fetuses delivered by elective Cesarean section to minimize potential variation in GR expression associated with labor.
Immunohistochemistry and GR antibodies
Paraffin-embedded blocks from nine human placenta pregnancies were identified from archived specimens from the Departments of Pathology, Weill Medical College of Cornell University and New York University School of Medicine. Institutional Review Board approval was obtained at both institutions. Immunohistochemical analysis was performed using an indirect immunoperoxidase method to identify total and phosphorylated GR. The tissues had been previously fixed in either Bouin’s solution or a solution containing 10% formalin for at least 2 h at room temperature, dehydrated through ethanol, cleared in chloroform, and embedded in paraffin. Five-micrometer tissue sections were serially cut on a microtome and mounted on glass slides. The sections were dewaxed overnight in an oven at 59 C followed by xylene, rehydrated, and washed in Tris-buffered saline (pH 7.4). For antigen retrieval, the tissue sections were heated in a microwave oven for 10 min (900 W, high power) three times in Target Retrieval solution (Dako Corp., Carpinteria, CA), cooled, and treated with 30% H2O2 for 30 min, rinsed with water, and blocked with 20% normal goat serum for 30 min. Serial sections were incubated overnight at 4 C with antibodies for total GR, GR-211P, and GR-203P (1:500 dilution) in 20% normal goat serum.
Antibodies had been previously generated against phosphopeptide sequences for hGR at the respective serine residues (27). The sera from the immunized rabbits had also been previously confirmed for specificity to phosphorylated GR by immunoblotting to cell lysates expressing wild-type hGR vs. mutations of hGR at those specific sites. The antibody for total GR (Ab218) was generated against the following peptide 194LQDLEFSSGSPO4PGKE207 and shown to react with GR independent of phosphorylation (Wang, Z., and M. Garabedian, unpublished observation). A second total GR rabbit polyclonal antibody (GR N499) raised against the first 499 amino acids of the hGR has been described previously and was used for immunoprecipitation and immunoblotting experiments (35). Antibodies for cytokeratin 7 (clone TL 12/30, M7018; Dako), common leukocyte marker CD45RB (clone PD7/26, M0833; Dako), -smooth muscle actin (A2547, Sigma-Aldrich, St. Louis, MO), and vimentin (clone V9, N1421; Dako) were used to identify placental cell types. A rabbit biotinylated secondary antibody/avidin-biotin complex was added to slides, which were then developed using diaminobenzidine substrate according to instructions in the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). The samples were counterstained with hematoxylin for nuclear localization.
Immunoreactivity was scored by a single examiner (H.Y.), an experienced immunohistopathologist who was blinded to the GR antibody used for the staining. The percentage of positive-staining cells were counted, regardless of intensity, and assessed by cell type per x200 field per slide studied. The extent of distribution of positive-staining cells was scored on a four-point scale for each placental cell type (0, no positive cells; 1+, 1–25% cells positive; 2+, 26–50% cells positive; 3+, 51–75% cells positive; and 4+, 76–100% cells positive), as previously described (36). An average of five random representative fields per slide for each placental specimen was analyzed. The cell types analyzed in the study included the following: SCT, CT, FIB, perivascular smooth muscle, and endothelial cells.
Cell culture
Fresh human placental tissue (n = 8) was collected prospectively from unidentified patients with the approval of the Institutional Review Board Associates at the New York University School of Medicine and Human Subjects Investigation Committee at Yale School of Medicine.
Placental FIB were isolated based on our previous method (3), which is a modification of methodology originally developed by Fant and Nanu (37). Five to 10 g of villous tissue were minced, washed with saline, and digested for 45 min in a 1:1 mixture of phenol red-free Ham’s F12-DMEM (basal medium) containing 0.1% collagenase and 0.01% DNAase. Dispersed cells were filtered through a 160-μm stainless-steel sieve and centrifuged (500 x g, 5 min). Cells were then resuspended in basal medium supplemented with 10% fetal bovine serum (FBS) medium and seeded in two T-75 culture flasks. On reaching 80% confluency, cells were subcultured in FBS medium. For experiments, FIB between passage 3–10 were plated in medium containing 10% charcoal-stripped FBS at a density of 5 x 105 cells in a 10-cm Petri dish. After 2–3 d, at approximately 80% confluency, FIB were treated with 100 nM dexamethasone (DEX) for the indicated time. Immunocytochemistry was performed on FIB grown on chamber slides (Lab-tek, Naperville, IL), which demonstrated that more than 99% of these cells were vimentin positive and confirmed their stromal origin (Fig. 1C, inset).
CT were isolated from approximately 45 g of human villous tissue at term after trypsin digestion and centrifugation on Percoll gradients. We have previously employed this procedure (38), which is a modification of those developed by Kliman et al. (39) and Douglas and King (40). Briefly, fresh placental tissue fragments were finely chopped, washed with saline, and treated with trypsin and DNAase. The digestate was poured through cheesecloth and two wire-mesh sieves with 0.0038- and 0.0021-in. openings. The effluent was collected and centrifuged at 500 x g for 5 min. The cell pellets were resuspended for centrifugation on a continuous Percoll gradient. The CT sedimenting, as a ring of cells at a density of approximately 1.05 g/ml, were washed and resuspended in basal medium supplemented with 10% heat-inactivated fetal calf serum (FCS) (Hyclone Laboratories, Logan, UT), 2 mM L-glutamine, 50 μg/ml penicillin, and 50 μg/ml streptomycin (Cellgro, Herndon, VA), i.e. FCS medium. The yield was approximately 3 x 108 cells per 45 g of villous tissue.
SCT were generated by culturing CT for 72 h in FCS medium. Under these conditions, the CT spontaneously differentiate into SCT, as previously described by Kliman et al. (39). The FIB, CT, and SCT were maintained at 37 C in FCS medium in a humidified atmosphere of 5% CO2-95% air.
Western blotting and immunoprecipitation
Extracts for immunoblotting were prepared from subconfluent 10-cm plates of FIB, CT, and SCT. Cells were maintained in 10% FCS medium, in which the serum was initially treated with dextran-coated charcoal to remove endogenous steroids. The cultures of CT, SCT, and FIB were treated with 100 nM DEX for the indicated time. Untreated CT, SCT, FIB were also lysed to quantify basal levels of GR protein expression. The cells were placed on ice, washed twice with phosphate-buffered saline (PBS), and lysed in 0.2 ml of buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 1% Triton X-100, 10% glycerol, and additional protease and phosphatase inhibitors [1 mM phenylmethylsulfonyl fluoride, 20 mM -glycerophosphate, 8 mM sodium pyrophosphate, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, and 1 μg/ml aprotinin (Roche Molecular Biochemicals, Indianapolis, IN)]. The lysates were centrifuged at 12,000 rpm for 15 min at 4 C. The soluble supernatants were normalized for total protein concentration using the Bio-Rad (Hercules, CA) protein assay. Samples were then boiled for 3 min in 2x sodium dodecyl sulfate sample buffer and stored at –20 C.
For Western blotting, approximately 20 μg of cell protein was subjected to electrophoresis using 7.5% Tris-HCl polyacrylamide gels or 4–15% Tris-HCl polyacrylamide gradient gels (Bio-Rad) in the presence of the reducing agent dithiothreitol (50 μM). Proteins were then transferred to Immobilon paper (Millipore Corp., Bedford, MA) at 110 V for 80 min. After transfer, blots were incubated with primary antibody at 4 C overnight at their respective dilutions: total GR (N499), 1:1,000; rabbit anti-hGR-S203P, 1:10,000; rabbit anti-hGR-S211P, 1:1,000; GR (no. PA3-514, Affinity BioReagents, Golden, CO), 1:500; mouse antihuman -actin (clone AC-15, Sigma, St. Louis, MO), 1:10,000; and mouse antihuman heat-shock protein (HSP) 90 (clone 68, BD Transduction Laboratories, San Jose, CA), 1:1,000. To detect GR, membranes were incubated for 1 h at room temperature with 0.2 μg/ml protein A conjugated to horseradish peroxidase (Kirkegaard & Perry Laboratories, Gaithersburg, MD). To detect -actin and HSP90, the blots were incubated with a 1:5,000 dilution of goat antimouse horseradish peroxidase conjugates. Blots were developed using enhanced chemiluminescence procedures according to the instructions of the manufacturer (Amersham Biosciences, Piscataway, NJ).
For immunoprecipitation studies, 10 μl of anti-GR antiserum (N499) were prebound to protein A/G Plus-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) in PBS at 4 C for 1.5 h and washed with PBS twice to remove the unbound antibody. The antibody-coated beads were incubated overnight at 4 C on a rotator with 1 mg of protein extract from either FIB, CT, or SCT cultures. The beads were then washed five times with PBS and twice with 50 mM Tris (pH 7.5), boiled in 2x sodium dodecyl sulfate sample buffer, and stored at –20 C. Western blotting and immunodetection were carried out as described above using the respective dilutions of total GR and phospho-GR antibodies.
For Western blotting results, intensities of bands on scanned autoradiographs were analyzed using National Institutes of Health Image J software (http://rsb.info.nih.gov/ij). Levels of total GR expression were obtained from normalizing the intensity of the GR band at approximately 97 kDa to that of HSP90 (Fig. 3) or -actin (Fig. 5). For Fig. 5, in which time-dependent down-regulation of GR by DEX treatment was studied, -actin-normalized levels of GR in control and DEX-treated cells were expressed relative to levels at time zero (T0). The effect of DEX treatment on phospho-GR expression (Fig. 3) is expressed as a percentage of control value. Quantitation of all Western blot results was performed for four independent experiments with cells obtained from four different placentas.
Real-time PCR
Real-time PCR was carried out essentially as we have previously described (3). Initially, Tri Reagent (Sigma) was used to extract RNA. Before use in real-time PCR, 2.5 μg of total RNA were reverse-transcribed in a 20 μl reaction containing 50 ng of random hexamers, 0.5 mM of each dNTP, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 5 mM MgCl2, 10 mM dithiothreitol, 40 U of RNase inhibitor, and 50 U of SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). Control reactions were set up without reverse transcriptase to assess the level of contaminating genomic DNA. All reactions were carried out at 25 C for 10 min, 42 C for 50 min, and 70 C for 15 min. The RNA template was removed from the cDNA:RNA hybrid by incubation with 2 U of RNase H at 37 C for 20 min. Amplification was performed on an iCycler iQ real-time PCR detection system (Bio-Rad). Primers for amplification of cDNAs were designed using Beacon Designer software from GenBank cDNA sequences, and the uniqueness of primers was established using BLAST search analysis. The following primers were used: GR (forward, 5'-CCTAAGGACGGTCTGAAGAGC-3'; (478 bp) reverse, 5'-GCCAAGTCTTGGCCCTCTAT-3'); -actin (forward, 5'-TTGGCAATGAGCGGTTCC-3'; (148 bp) reverse, 5'-AGCACTGTGTTGGCGTAC-3'); and 18S rRNA. GR and -actin primers were purchased from Invitrogen. The proprietary 18S rRNA primers from Ambion (QuantumRNA Universal 18S Internal Standard, catalog no. 1718; Austin, TX) were used to amplify this control rRNA sequence. Aliquots containing 25 ng of cDNA were amplified in a total volume of 25 μl containing 12.5 μl of a 2x iQ SYBR Green Supermix and 0.5 μM each primer. All samples were run in triplicate. The reactions were heated at 95 C for 3 min to activate Taq polymerase followed by 40 cycles of 95 C for 10 sec, 56 C for 30 sec (annealing temperature) and 72 C for 30 sec. Melting curves of the products were obtained after cycling by a stepwise increase of temperature from 55–95 C. PCR products were electrophoresed on agarose gels to confirm the presence of a single product of the expected size. Amplified products were sequenced to verify their identity. For the quantification of relative levels of gene expression, the comparative threshold cycle (CT) method was employed as described in User Bulletin No. 2 for ABI PRISM 7700 Sequence Detection System. CT represents the PCR cycle at which an increase in reporter fluorescence above a background signal can first be detected (10 times the SD of the baseline). The 18S rRNA or -actin CT values were subtracted from GR CT values to derive a CT value. The relative expression of the gene of interest was then evaluated using the expression 2–CT. The value for CT was obtained by subtracting the CT of the calibrator (i.e. the 24-h control value within each experimental run) from each CT in that experiment. Cumulative 2–CT results are presented as a mean ± SE obtained in three independent experiments from three different placentas.
Statistics
Results are expressed as a mean ± SE. Data were analyzed by ANOVA or Student’s t test using SigmaStat software from Jandel Scientific (San Rafael, CA). A value of P < 0.05 was considered significant.
Results
Expression of GR in human term placenta
Before analyzing the expression of GR in the placenta, we determined the integrity of the tissue sections by hematoxylin and eosin staining (Fig. 1A). In addition, we established the distribution of cell types within the placental villus by immunohistochemistry using antibodies against cytokeratin 7 to identify cells of trophoblast origin (Fig. 1A, inset), CD45RB to locate immune cells (Fig. 1B), and vimentin to distinguish FIB (Fig. 1C). CT were found as a discontinuous cell layer beneath the SCT (Fig. 1A, inset), whereas FIB were observed throughout the villous core (Fig. 1C). In addition, we observed more than 99% vimentin staining of cultured placental FIB by immunocytochemistry (Fig. 1C, inset). Immune cells, both maternal and fetal, were occasionally observed (Fig. 1B). Having established the cell-type distribution within the placental architecture, we examined GR expression in the human placenta by immunohistochemistry. We observed strong GR immunoreactivity (brown-staining cells) within the nuclei of FIB in the mesenchyme of terminal villi (Fig. 1D). In contrast, CT showed a lower level of GR immunoreactivity in nuclei relative to the FIB, and even lower levels of GR immunoreactivity were noted in nuclei and cytoplasm of SCT (blue-staining cells) (Fig. 1D). Very few macrophages or immune cells were detected in the villous core; the majority of GR-positive cells in the villus were of trophoblast and FIB origin. No staining was observed above background when preimmune serum was employed (Fig. 1D, inset). Thus, the steady-state level of GR differs among FIB, CT, and SCT.
We also examined which cell types exhibit the phosphorylated forms of GR by immunohistochemistry using GR phosphorylation site-specific antibodies to S203 and S211. GR phosphorylation at these sites is indicative of various states of receptor activity. For example, phosphorylation of GR at S211 is strictly agonist-dependent, and positive staining with the GR-S211P antibody is a surrogate marker for this activated form of GR. GR-S203P reactivity was primarily localized to the nuclei of FIB within the villous core, whereas little GR-S203P immunoreactivity was observed in the trophoblast layer (Fig. 1E). The GR-S211P antibody reacted with a majority of the nuclei of placental FIB but with only a minority of nuclei in trophoblasts (Fig. 1F). Again, no staining above background was observed when preimmune sera were employed (Fig. 1, E and F, insets). The results from the immunohistochemical analysis of total GR and phospho-GR expression are summarized in Table 1. In addition to differences in total GR expression, our findings reveal distinct patterns of GR phosphorylation among placental cell types indicative of differences in cell-signaling pathways and GR activation status in vivo. GR appears to be more transcriptionally active in FIB compared with SCT of the term human placenta.
To confirm the cell type-specific pattern of GR expression determined by immunohistochemistry, we cultured primary human placental FIB, CT, and SCT and analyzed GR expression from cell lysates by immunoblotting (Fig. 2A). Note that signal for GR was detected at molecular masses of approximately 97 kDa (denoted by arrow) and 92 kDa in FIB. The 97-kDa band corresponds to the published molecular mass of GR (41), whereas the 92-kDa band was presumed to reflect GR (41). However, Western blotting with an anti-GR antibody did not consistently detect a 92-kDa protein in cell extracts, suggesting that this band is nonspecific. Quantification of GR signals normalized to that of HSP90 revealed greater levels of GR in FIB compared with CT or SCT (Fig. 2B). This result is consistent with our immunohistochemical data demonstrating that patterns of GR expression are faithfully maintained in cultured cells.
We also examined cultured cells for changes in GR phosphorylation as a function of GC treatment (Fig. 3A). In FIB, the GR-S203P antibody recognized GR from both untreated and hormone-treated FIB, and the same pattern was discernible in CT, albeit at a weaker signal intensity. No signal was detected in SCT, most likely due to very low level expression of GR in SCT. The GR-S211P antibody, which showed very weak immunoreactivity toward GR from untreated FIB, displayed substantially more immunoreactivity toward GR from DEX-treated cells. Again, a similar pattern was noted in CT. Based on the relatively high levels of GR expression in FIB, we were able to quantify the effects of DEX treatment on phospho-GR expression in these cells. We noted that DEX treatment significantly enhanced GR-S211 phosphorylation by 2.2-fold compared with control, whereas although the effects on GR-S203 were less pronounced, they also reached statistical significance (Fig. 3B). Thus, GR phosphorylation at S211 is clearly hormone dependent in FIB and is consistent with an important role of phosphorylation and transcriptional activity of GR after GC exposure in this cell type.
We next examined the expression of GR and phospho-GR in the larger stem villi of the placenta by immunohistochemistry to further characterize GR expression in major placental vasculature. FIB were again identified primarily by vimentin staining of the cytoplasm (Fig. 4F), smooth muscle cells were confirmed by expression of -smooth muscle actin (Fig. 4D), and immune cells were located by CD45 staining (Fig. 4E). Immune macrophages were rarely found in the placental mesenchymal core. We found that expression of total GR and GR-S211P were confined to the nuclei of perivascular smooth muscle cells and FIB throughout the core of the villus (Fig. 4, A and C). A low level of GR-S203P staining was noted only in the nuclei of vascular smooth muscle cells (Fig. 4B). Endothelial cells showed low or variable levels of staining for total GR and phospho-GRs relative to smooth muscle cells and FIB. Thus, differentially phosphorylated receptor species are located in specific cell types of the placenta, which likely represent distinct aspects of receptor function in vivo.
Effect of DEX treatment on GR levels in placental FIB
To examine the effect of GC treatment on the level of GR protein in FIB, cells were maintained in medium with or without 100 nM DEX for 7, 24, and 48 h, respectively. Lysates were prepared, and the expression of GR was examined by immunoblotting with an antibody against total GR (Fig. 5A). Levels of GR at T0 and in control and DEX-treated samples were normalized to that of -actin by densitometric analysis of autoradiographs. GR levels in control and DEX-treated cells were then expressed as a percentage of the T0 value. No significant time-dependent change in GR levels for control cells was noted (Fig. 5B). In contrast, DEX treatment reduced GR levels to 27 ± 3, 17 ± 3, and 11 ± 3% of control levels at 7, 24, and 96 h, respectively (Fig. 5A). Thus, GR undergoes rapid hormone-dependent down-regulation in placental FIB.
Effect of DEX treatment on levels of GR mRNA in placental FIB
Previous studies indicated that GC treatment may suppress GR expression through changes in transcription and/or stability of GR protein (31, 33, 34, 42). We used real-time PCR analysis to determine the effects of GC treatment on levels of GR mRNA in FIB. We observed that treatment of FIB with 100 nM DEX for 24 or 48 h did not significantly affect the expression of GR mRNA when normalized to levels of 18S or -actin mRNA (Fig. 6). This suggests that DEX treatment reduced GR expression in FIB through changes in receptor protein stability.
Discussion
GC act via the GR to govern cell differentiation, apoptosis, and response to environmental stresses (43, 44). GR is a transcription factor that is expressed in virtually every mammalian cell, but GC-responsive genes are regulated in a tissue- and cell type-specific manner. Previous studies have demonstrated that GR is expressed in human placenta (19, 20, 21) and that GC treatment alters gene expression in cultures of trophoblasts and FIB isolated from human placentas (2, 3, 7, 38). However, little is known concerning cell type-specific expression of GR and its regulation in human placenta. Our current study clearly demonstrates that FIB in the villous core of the human placenta are targets of GC action. These cells are of critical importance in maintaining placental architecture because ECM proteins synthesized by FIB provide a biological support for CT and SCT layers on the surface of the villus and fetal capillaries below (9, 10, 11, 12). Our initial results using immunohistochemical methods showed that placental FIB express relatively high levels of GR and phospho-GR compared with trophoblast and endothelial cells in terminal villi. Of note was our observation of high levels of GR-S211P in FIB. We previously reported that phosphorylation of GR on S211 was associated with nuclear localization and transcriptional activation of GR after hormone treatment (27). This suggests that placental FIB express transcriptionally active phospho-GR in vivo. In support of this are in vitro studies that showed that DEX treatment enhanced phosphorylation of S211 on the GR of FIB. Of note, Western blotting detected not only the GR species at the predicted molecular mass of 97 kDa (41) (Fig. 2A), but also a 92-kDa protein, the molecular mass reported for GR (41). Our results suggest that the protein detected in our Western blots is most likely not GR because GR antibody did not consistently detect a 92-kDa species (not shown). In addition, the N499 antibody used for immunoprecipitation studies, which recognizes an epitope present in both GR and GR, did not detect the 92-kDa species in these experiments (Fig. 3A).
Our findings from immunohistochemistry and immunoblotting studies using total and phospho-GR antibodies provides insight into how GR is distributed throughout the human placenta and demonstrates how the major placental cell types are differentially phosphorylated. There appears to be a distinct pattern of GR phosphorylation in the placental villus by cell type, which suggests that activation of the GR in each specific cell type may result in selective regulation of cell-specific GR target genes (25). In other words, we suspect that the consistent variation in levels of GR-S203P and GR-S211P in each particular cell type reflects a particular sensitivity to GC treatment and transcriptional activation of specific GR target genes. These results support our previous studies demonstrating GC responsiveness of placental FIB because hormone treatment was found to markedly increase ECM protein expression in FIB isolated from human and baboon placentas (7), whereas other authors have demonstrated that the lung / hydrolase gene is uniquely induced in the lung epithelium A549 cell line (45). Based on our observations that FIB are GC responsive, it is not surprising that the number of doses of antenatal GC given to women at risk for preterm delivery to enhance fetal lung maturation was directly correlated with the severity of villous fibrosis in the placenta (46).
The finding that FIB are targets of GC action is not surprising because GC-regulated genes, including the ECM proteins fibronectin and collagen, are synthesized by placental FIB (3, 12). However, two other GC-regulated genes, CRH and hCG, are synthesized by SCT (4, 5, 47), a cell type shown in the present study to express relatively low levels of GR. This suggests that GC effects in SCT may be indirect or that the low level of GR in SCT is sufficient to promote GC-dependent changes in gene expression. It is of note in the current study that we used a concentration of DEX of 100 nM to elicit GC-dependent responses in placental cells. This concentration of DEX has been previously shown to promote maximal changes in ECM, CRH, and hCG expression in placental and amnion cells by our group and others (3, 4, 5, 7). In addition, 100 nM was the dose of DEX previously used to demonstrate GC-mediated down-regulation of GR (31, 33, 34, 42). The observation that levels of cortisol in maternal sera range from approximately 200–400 nM between 8 wk of pregnancy and term indicates that periplacental levels of GC are high throughout gestation (48).
In the current study, we also report that DEX treatment reduces GR protein expression in FIB between 7 and 48 h of treatment. This phenomenon, known as homologous down-regulation, has been observed in several cell types and has been suggested to represent a mechanism through which cells limit GC responsiveness (31, 32). It is generally thought that homologous down-regulation occurs through cell type-specific mechanisms involving either reduced GR transcription or protein stability and that changes in GR mRNA stability do not play a major role in this process (32). GC-mediated suppression of GR levels has been demonstrated to involve ubiquitination and proteasome-mediated degradation of GR in several cell types (31, 49). Our results suggest that GC-mediated down-regulation of GR levels occurs through changes in protein stability because we observed no change in GR mRNA levels in FIB after DEX treatment.
In conclusion, our studies clearly demonstrate that FIB in the core of the placental villus are a major target of GC action. Further studies will be needed to elucidate how GC and GR interact in the human placenta to determine their role in placental pathophysiology.
Acknowledgments
We are grateful to Dr. Inez Pineda-Torra for critically reading the manuscript.
Footnotes
This work was supported, in part, by National Institutes of Health Grants DK54836 (to M.J.G.) and HD33909 (to S.G.). M.-J.L. is the recipient of a Reproductive Scientist Development Program/Wyeth Award (National Institutes of Health Grant 5K12HD00849).
Abbreviations: CT, Cytotrophoblast(s); CT, threshold cycle; DEX, dexamethasone; ECM, extracellular matrix; FBS, fetal bovine serum; FCS, fetal calf serum; FIB, fibroblast(s); GC, glucocorticoid; GR, glucocorticoid receptor; hCG, human chorionic gonadotropin; hGR, human glucocorticoid receptor; HSP, heat-shock protein; IUGR, intrauterine growth restriction; PBS, phosphate-buffered saline; SCT, syncytiotrophoblast(s); T0, time zero.
References
Guller S, Wozniak R, Krikun G, Burnham JM, Kaplan P, Lockwood CJ 1993 Glucocorticoid suppression of human placental fibronectin expression: implications in uterine-placental adherence. Endocrinology 133:1139–1146
Ryu JS, Majeska RJ, Ma Y, LaChapelle L, Guller S 1999 Steroid regulation of human placental integrins: suppression of 2 integrin expression in cytotrophoblasts by glucocorticoids. Endocrinology 140:3904–3908
Lee MJ, Ma Y, LaChapelle L, Kadner SS, Guller S 2004 Glucocorticoid enhances transforming growth factor- effects on extracellular matrix protein expression in human placental mesenchymal cells. Biol Reprod 70:1246–1252
Ringler GE, Kallen CB, Strauss 3rd JF 1989 Regulation of human trophoblast function by glucocorticoids: dexamethasone promotes increased secretion of chorionic gonadotropin. Endocrinology 124:1625–1631
Jones SA, Brooks AN, Challis JR 1989 Steroids modulate corticotropin-releasing hormone production in human fetal membranes and placenta. J Clin Endocrinol Metab 68:825–830
Ma Y, Ryu JS, Dulay A, Segal M, Guller S 2002 Regulation of plasminogen activator inhibitor (PAI)-1 expression in a human trophoblast cell line by glucocorticoid (GC) and transforming growth factor (TGF)-. Placenta 23:727–734
Ma Y, Lockwood CJ, Bunim AL, Giussani DA, Nathanielsz PW, Guller S 2000 Cell type-specific regulation of fetal fibronectin expression in amnion: conservation of glucocorticoid responsiveness in human and nonhuman primates. Biol Reprod 62:1812–1817
Hahn T, Barth S, Graf R, Engelmann M, Beslagic D, Reul JM, Holsboer F, Dohr G, Desoye G 1999 Placental glucose transporter expression is regulated by glucocorticoids. J Clin Endocrinol Metab 84:1445–1452
Benirschke K, Kaufmann P, eds. 2000 Placental types. In: Pathology of the human placenta. 4th ed. New York: Springer-Verlag; 29–41
Amenta PS, Gay S, Vaheri A, Martinez-Hernandez A 1986 The extracellular matrix is an integrated unit: ultrastructural localization of collagen types I, III, IV, V, VI, fibronectin, and laminin in human term placenta. Coll Relat Res 6:125–152
Yamada T, Isemura M, Yamaguchi Y, Munakata H, Hayashi N, Kyogoku M 1987 Immunohistochemical localization of fibronectin in the human placentas at their different stages of maturation. Histochemistry 86:579–584
Chen CP, Aplin JD 2003 Placental extracellular matrix: gene expression, deposition by placental fibroblasts and the effect of oxygen. Placenta 24:316–325
Benirschke K, Kaufmann P, eds. 2000 Architecture of normal villous trees. In: Pathology of the human placenta. 4th ed. New York: Springer-Verlag; 116–154
Macara L, Kingdom JC, Kaufmann P, Kohnen G, Hair J, More IA, Lyall F, Greer IA 1996 Structural analysis of placental terminal villi from growth-restricted pregnancies with abnormal umbilical artery Doppler waveforms. Placenta 17:37–48
Salafia CM, Minior VK, Pezzullo JC, Popek EJ, Rosenkrantz TS, Vintzileos AM 1995 Intrauterine growth restriction in infants of less than thirty-two weeks’ gestation: associated placental pathologic features. Am J Obstet Gynecol 173:1049–1057
Heckmann M, Wudy SA, Haack D, Pohlandt F 1999 Reference range for serum cortisol in well preterm infants. Arch Dis Child Fetal Neonatal Ed 81:F171–F174
Parker Jr CR, Buchina ES, Barefoot TK 1994 Abnormal adrenal steroidogenesis in growth-retarded newborn infants. Pediatr Res 35:633–636
Seckl JR 1997 Glucocorticoids, feto-placental 11-hydroxysteroid dehydrogenase type 2, and the early life origins of adult disease. Steroids 62:89–94
Driver PM, Kilby MD, Bujalska I, Walker EA, Hewison M, Stewart PM 2001 Expression of 11-hydroxysteroid dehydrogenase isozymes and corticosteroid hormone receptors in primary cultures of human trophoblast and placental bed biopsies. Mol Hum Reprod 7:357–363
Patel FA, Funder JW, Challis JR 2003 Mechanism of cortisol/progesterone antagonism in the regulation of 15-hydroxyprostaglandin dehydrogenase activity and messenger ribonucleic acid levels in human chorion and placental trophoblast cells at term. J Clin Endocrinol Metab 88:2922–2933
Chan CC, Lao TT, Ho PC, Sung EO, Cheung AN 2003 The effect of mifepristone on the expression of steroid hormone receptors in human decidua and placenta: a randomized placebo-controlled double-blind study. J Clin Endocrinol Metab 88:5846–5850
Yamamoto KR 1985 Steroid receptor regulated transcription of specific genes and gene networks. Annu Rev Genet 19:209–252
Hollenberg SM, Weinberger C, Ong ES, Rosenfeld MG, Evans RM 1985 Primary structure and expression of a functional human glucocorticoid receptor cDNA. Nature 318:635–641
Bamberger CM, Bamberger AM, de Castro M, Chrousos GP 1995 Glucocorticoid receptor , a potential endogenous inhibitor of glucocorticoid action in humans. J Clin Invest 95:2435–2441
Ismaili N, Garabedian MJ 2004 Modulation of glucocorticoid receptor function via phosphorylation. Ann NY Acad Sci 1024:86–101
Krstic MD, Rogatsky I, Yamamoto KR, Garabedian MJ 1997 Mitogen-activated and cyclin-dependent protein kinases selectively and differentially modulate transcriptional enhancement by the glucocorticoid receptor. Mol Cell Biol 17:3947–3954
Wang Z, Frederick J, Garabedian MJ 2002 Deciphering the phosphorylation "code" of the glucocorticoid receptor in vivo. J Biol Chem 277:26573–26580
Cidlowski JA, Cidlowski NB 1981 Regulation of glucocorticoid receptors by glucocorticoids in cultured HeLa S3 cells. Endocrinology 109:1975–1982
Svec F, Rudis M 1981 Glucocorticoids regulate the glucocorticoid receptor in the AtT-20 cell. J Biol Chem 256:5984–5987
Lacroix A, Bonnard GD, Lippman ME 1984 Modulation of glucocorticoid receptors by mitogenic stimuli, glucocorticoids and retinoids in normal human cultured T cells. J Steroid Biochem 21:73–80
Wallace AD, Cidlowski JA 2001 Proteasome-mediated glucocorticoid receptor degradation restricts transcriptional signaling by glucocorticoids. J Biol Chem 276:42714–42721
Schaaf MJ, Cidlowski JA 2002 Molecular mechanisms of glucocorticoid action and resistance. J Steroid Biochem Mol Biol 83:37–48
Dong Y, Poellinger L, Gustafsson JA, Okret S 1988 Regulation of glucocorticoid receptor expression: evidence for transcriptional and posttranslational mechanisms. Mol Endocrinol 2:1256–1264
McIntyre WR, Samuels HH 1985 Triamcinolone acetonide regulates glucocorticoid-receptor levels by decreasing the half-life of the activated nuclear-receptor form. J Biol Chem 260:418–427
Rogatsky I, Zarember KA, Yamamoto KR 2001 Factor recruitment and TIF2/GRIP1 corepressor activity at a collagenase-3 response element that mediates regulation by phorbol esters and hormones. EMBO J 20:6071–6083
Tassone P, Tuccillo F, Bonelli P, D’Armiento FP, Bond HM, Palmieri C, Tagliaferri P, Venuta S 2002 Fetal ontogeny and tumor expression of the early thymic antigen UN1. Int J Oncol 20:707–711
Fant ME, Nanu L 1995 Human placental endothelin: expression of endothelin-1 mRNA by human placental fibroblasts in culture. Mol Cell Endocrinol 109:119–123
Guller S, LaCroix NC, Kirkun G, Wozniak R, Markiewicz L, Wang EY, Kaplan P, Lockwood CJ 1993 Steroid regulation of oncofetal fibronectin expression in human cytotrophoblasts. J Steroid Biochem Mol Biol 46:1–10
Kliman HJ, Nestler JE, Sermasi E, Sanger JM, Strauss 3rd JF 1986 Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 118:1567–1582
Douglas GC, King BF 1989 Isolation of pure villous cytotrophoblast from term human placenta using immunomagnetic microspheres. J Immunol Methods 119:259–268
Gupta S, Gyomorey S, Lye SJ, Gibb W, Challis JR 2003 Effect of labor on glucocorticoid receptor (GR(Total), GR, and GR) proteins in ovine intrauterine tissues. J Soc Gynecol Investig 10:136–144
Rosewicz S, McDonald AR, Maddux BA, Goldfine ID, Miesfeld RL, Logsdon CD 1988 Mechanism of glucocorticoid receptor down-regulation by glucocorticoids. J Biol Chem 263:2581–2584
Munck A, Mendel DB, Smith LI, Orti E 1990 Glucocorticoid receptors and actions. Am Rev Respir Dis 141:S2–S10
Sapolsky RM, Romero LM, Munck AU 2000 How do glucocorticoids influence stress responses Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr Rev 21:55–89
Rogatsky I, Wang JC, Derynck MK, Nonaka DF, Khodabakhsh DB, Haqq CM, Darimont BD, Garabedian MJ, Yamamoto KR. 2003 Target-specific utilization of transcriptional regulatory surfaces by the glucocorticoid receptor. Proc Natl Acad Sci USA 100:13845–13850
Ghidini, A, Pezzullo JC, Sylvestre G, Lembet A, Salafia CM 2001 Antenatal corticosteroids and placental histology in preterm birth. Placenta 22:412–417
Riley SC, Walton JC, Herlick JM, Challis JR 1991 The localization of corticotropin-releasing hormone in the human placenta and fetal membranes throughout gestation. J Clin Endocrinol Metab 72:1001–1007
Dorr HG, Heller A, Versmold HT, Sippell WG, Herrmann M, Bidlingmaier F, Knorr D 1989 Longitudinal study of progestins, mineralocorticoids, and glucocorticoids throughout human pregnancy. J Clin Endocrinol Metab 68:863–868
Sengupta S, Wasylyk B 2001 Ligand-dependent interaction of the glucocorticoid receptor with p53 enhances their degradation by Hdm2. Genes Dev 15:2367–2380(Men-Jean Lee, Zhen Wang, )
Department of Obstetrics and Gynecology (Y.M., L.Y., S.G.), Yale University School of Medicine, New Haven, Connecticut 06520-8063
Department of Pathology (R.N.B.), Weill Medical College of Cornell University, New York, New York 10021
Abstract
The human placenta is a glucocorticoid (GC)-responsive organ consisting of multiple cell types including smooth muscle cells, fibroblasts, and trophoblast that demonstrate changes in gene expression after hormone treatment. However, little is known about the relative expression or activity of the GC receptor (GR) among the various placental cell types. Normal term human placentas were examined by immunohistochemistry using either GR phosphorylation site-specific antibodies that are markers for various activation states of the GR or a GR antibody that recognizes the receptor independent of its phosphorylation state (total GR). We found strong total GR and phospho-GR immunoreactivity in stromal fibroblasts of terminal villi, as well as perivascular fibroblasts and vascular smooth muscle cells of the stem villi. Lower levels of both total GR and phospho-GR were found within cytotrophoblast cells relative to fibroblasts, whereas syncytiotrophoblast showed very little total GR or phospho-GR immunoreactivity. This pattern holds true for immunoblot analysis of extracts from cell fractions cultured ex vivo. In cultured placental fibroblasts, phosphorylation of GR increased upon short-term GC treatment, consistent with a role for GR phosphorylation in receptor transactivation. Total GR levels were reduced by nearly 90% after long-term hormone treatment; however, this down-regulation was independent of changes in GR mRNA levels. These findings demonstrate that GR levels in fibroblasts can be modulated by changes in hormone exposure. Such cell type-specific differences in GR protein expression and phosphorylation may provide the means of differentially regulating the GC response among the cells of the human placenta.
Introduction
PREVIOUS STUDIES BY our laboratory and others (1, 2, 3) have documented that glucocorticoid (GC) is a key regulator of placental gene expression. GC-regulated genes in placenta include human chorionic gonadotropin (hCG) (4), CRH (5), plasminogen activator inhibitor-1 (6), fibronectin (7), and glucose transporters (8), demonstrating that the human placenta is a GC-responsive organ. Of particular relevance to the current study are the findings that GCs enhance the expression of extracellular matrix (ECM) proteins by placental fibroblasts (FIB) (i.e. stromal cells from the villous core of the placenta) in human and nonhuman primates (3, 7). The ECM synthesized by these placental cells plays an important role in maintaining tissue structure by providing a biological support for cytotrophoblast (CT) and syncytiotrophoblast (SCT) layers on the surface of the villus and fetal capillaries below (9, 10, 11, 12). FIB are present in both large stem villi, which form the main trunk of the villous tree (300 μm in diameter), and terminal villi, which are the final villous branches (50 μm in diameter) that are the main site of nutrient exchange between mother and fetus (13). Excessive synthesis of ECM proteins by placental FIB has been noted in pregnancies complicated by intrauterine growth restriction (IUGR) (14, 15). These pregnancies are also characterized by aberrantly high periplacental levels of GC (16, 17, 18), but the significance of this observation in the pathophysiology of IUGR remains to be elucidated.
Although several studies have examined GC-mediated gene regulation in human placenta (2, 3, 4, 5), little is known concerning the expression and regulation of placental GC receptor (GR). It has been demonstrated that human placenta expresses GR (19, 20, 21), but patterns of cell type-specific expression and regulation have not been established. GR is expressed in virtually every mammalian cell but controls distinct subsets of responsive genes, depending on cell type (22). The human GR (hGR) has two isoforms, GR and GR, that arise due to alternative splicing of the 3' terminus of the GR gene (23). The GR isoform was reported to be transcriptionally inactive and inhibited GR-mediated effects (24).
Besides being a ligand-dependent transcription factor, GR is also subject to posttranslational modification through phosphorylation (25). GR is phosphorylated in the absence of hormone, and additional phosphorylation events occur after agonist binding. Three major serine sites are phosphorylated within the N-terminal region of the receptor that are involved in transcriptional regulation (S203, S211, and S226 using the human numbering scheme). We have also identified the kinases that target these sites in vitro: cyclin E/Cdk2 and cyclin A/Cdk2 phosphorylate S203 and S211, respectively, whereas c-Jun N-terminal kinases phosphorylate S226 (26). GR phosphorylation may determine target gene activity, cofactor interaction, strength and duration of receptor signaling, or receptor stability. For example, phosphorylation at S211 has been associated with nuclear translocation and transcriptional activation of GR after hormone treatment (27). There is some evidence to suggest that phosphorylation at S203 is a determinant of ligand-dependent down-regulation of GR (27), but the exact mechanisms have not been clearly defined. These findings strongly suggest that changes in GR phosphorylation render the receptor differentially responsive to GC treatment. The phosphorylated isoforms of GR can be considered surrogates for various transcriptionally active states of the receptor (25). We have recently generated novel GR phosphorylation-specific antibodies to these phospho-serine sites to allow a detailed analysis by immunohistochemistry of GR phospho-isoform expression in vivo without the use of radioactivity (27).
In addition to the rapid (1–2-h) effects of GC on GR phosphorylation, it has been demonstrated that long-term (>4-h) treatment with GC promotes down-regulation of GR in several cell types (28, 29, 30). GC-dependent suppression of GR (i.e. homologous down-regulation) has been demonstrated to be a mechanism to limit hormone responsiveness (31, 32). Although not completely elucidated, the cellular mechanisms of homologous down-regulation include inhibition of GR transcription (15, 16) and decreased GR protein stability (21, 33, 34). These pathways also appear to be cell-type specific (32).
Therefore, the purpose of the present study was to establish the patterns of expression of total GR and phospho-GR in human placental tissue and then to examine their regulation in placental FIB in vitro.
Materials and Methods
Tissue procurement
Placental tissue used for immunohistochemical and cell-culture studies was obtained from uncomplicated pregnancies from normal term (36–41 wk gestation) with appropriately grown, singleton fetuses delivered by elective Cesarean section to minimize potential variation in GR expression associated with labor.
Immunohistochemistry and GR antibodies
Paraffin-embedded blocks from nine human placenta pregnancies were identified from archived specimens from the Departments of Pathology, Weill Medical College of Cornell University and New York University School of Medicine. Institutional Review Board approval was obtained at both institutions. Immunohistochemical analysis was performed using an indirect immunoperoxidase method to identify total and phosphorylated GR. The tissues had been previously fixed in either Bouin’s solution or a solution containing 10% formalin for at least 2 h at room temperature, dehydrated through ethanol, cleared in chloroform, and embedded in paraffin. Five-micrometer tissue sections were serially cut on a microtome and mounted on glass slides. The sections were dewaxed overnight in an oven at 59 C followed by xylene, rehydrated, and washed in Tris-buffered saline (pH 7.4). For antigen retrieval, the tissue sections were heated in a microwave oven for 10 min (900 W, high power) three times in Target Retrieval solution (Dako Corp., Carpinteria, CA), cooled, and treated with 30% H2O2 for 30 min, rinsed with water, and blocked with 20% normal goat serum for 30 min. Serial sections were incubated overnight at 4 C with antibodies for total GR, GR-211P, and GR-203P (1:500 dilution) in 20% normal goat serum.
Antibodies had been previously generated against phosphopeptide sequences for hGR at the respective serine residues (27). The sera from the immunized rabbits had also been previously confirmed for specificity to phosphorylated GR by immunoblotting to cell lysates expressing wild-type hGR vs. mutations of hGR at those specific sites. The antibody for total GR (Ab218) was generated against the following peptide 194LQDLEFSSGSPO4PGKE207 and shown to react with GR independent of phosphorylation (Wang, Z., and M. Garabedian, unpublished observation). A second total GR rabbit polyclonal antibody (GR N499) raised against the first 499 amino acids of the hGR has been described previously and was used for immunoprecipitation and immunoblotting experiments (35). Antibodies for cytokeratin 7 (clone TL 12/30, M7018; Dako), common leukocyte marker CD45RB (clone PD7/26, M0833; Dako), -smooth muscle actin (A2547, Sigma-Aldrich, St. Louis, MO), and vimentin (clone V9, N1421; Dako) were used to identify placental cell types. A rabbit biotinylated secondary antibody/avidin-biotin complex was added to slides, which were then developed using diaminobenzidine substrate according to instructions in the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). The samples were counterstained with hematoxylin for nuclear localization.
Immunoreactivity was scored by a single examiner (H.Y.), an experienced immunohistopathologist who was blinded to the GR antibody used for the staining. The percentage of positive-staining cells were counted, regardless of intensity, and assessed by cell type per x200 field per slide studied. The extent of distribution of positive-staining cells was scored on a four-point scale for each placental cell type (0, no positive cells; 1+, 1–25% cells positive; 2+, 26–50% cells positive; 3+, 51–75% cells positive; and 4+, 76–100% cells positive), as previously described (36). An average of five random representative fields per slide for each placental specimen was analyzed. The cell types analyzed in the study included the following: SCT, CT, FIB, perivascular smooth muscle, and endothelial cells.
Cell culture
Fresh human placental tissue (n = 8) was collected prospectively from unidentified patients with the approval of the Institutional Review Board Associates at the New York University School of Medicine and Human Subjects Investigation Committee at Yale School of Medicine.
Placental FIB were isolated based on our previous method (3), which is a modification of methodology originally developed by Fant and Nanu (37). Five to 10 g of villous tissue were minced, washed with saline, and digested for 45 min in a 1:1 mixture of phenol red-free Ham’s F12-DMEM (basal medium) containing 0.1% collagenase and 0.01% DNAase. Dispersed cells were filtered through a 160-μm stainless-steel sieve and centrifuged (500 x g, 5 min). Cells were then resuspended in basal medium supplemented with 10% fetal bovine serum (FBS) medium and seeded in two T-75 culture flasks. On reaching 80% confluency, cells were subcultured in FBS medium. For experiments, FIB between passage 3–10 were plated in medium containing 10% charcoal-stripped FBS at a density of 5 x 105 cells in a 10-cm Petri dish. After 2–3 d, at approximately 80% confluency, FIB were treated with 100 nM dexamethasone (DEX) for the indicated time. Immunocytochemistry was performed on FIB grown on chamber slides (Lab-tek, Naperville, IL), which demonstrated that more than 99% of these cells were vimentin positive and confirmed their stromal origin (Fig. 1C, inset).
CT were isolated from approximately 45 g of human villous tissue at term after trypsin digestion and centrifugation on Percoll gradients. We have previously employed this procedure (38), which is a modification of those developed by Kliman et al. (39) and Douglas and King (40). Briefly, fresh placental tissue fragments were finely chopped, washed with saline, and treated with trypsin and DNAase. The digestate was poured through cheesecloth and two wire-mesh sieves with 0.0038- and 0.0021-in. openings. The effluent was collected and centrifuged at 500 x g for 5 min. The cell pellets were resuspended for centrifugation on a continuous Percoll gradient. The CT sedimenting, as a ring of cells at a density of approximately 1.05 g/ml, were washed and resuspended in basal medium supplemented with 10% heat-inactivated fetal calf serum (FCS) (Hyclone Laboratories, Logan, UT), 2 mM L-glutamine, 50 μg/ml penicillin, and 50 μg/ml streptomycin (Cellgro, Herndon, VA), i.e. FCS medium. The yield was approximately 3 x 108 cells per 45 g of villous tissue.
SCT were generated by culturing CT for 72 h in FCS medium. Under these conditions, the CT spontaneously differentiate into SCT, as previously described by Kliman et al. (39). The FIB, CT, and SCT were maintained at 37 C in FCS medium in a humidified atmosphere of 5% CO2-95% air.
Western blotting and immunoprecipitation
Extracts for immunoblotting were prepared from subconfluent 10-cm plates of FIB, CT, and SCT. Cells were maintained in 10% FCS medium, in which the serum was initially treated with dextran-coated charcoal to remove endogenous steroids. The cultures of CT, SCT, and FIB were treated with 100 nM DEX for the indicated time. Untreated CT, SCT, FIB were also lysed to quantify basal levels of GR protein expression. The cells were placed on ice, washed twice with phosphate-buffered saline (PBS), and lysed in 0.2 ml of buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 1% Triton X-100, 10% glycerol, and additional protease and phosphatase inhibitors [1 mM phenylmethylsulfonyl fluoride, 20 mM -glycerophosphate, 8 mM sodium pyrophosphate, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, and 1 μg/ml aprotinin (Roche Molecular Biochemicals, Indianapolis, IN)]. The lysates were centrifuged at 12,000 rpm for 15 min at 4 C. The soluble supernatants were normalized for total protein concentration using the Bio-Rad (Hercules, CA) protein assay. Samples were then boiled for 3 min in 2x sodium dodecyl sulfate sample buffer and stored at –20 C.
For Western blotting, approximately 20 μg of cell protein was subjected to electrophoresis using 7.5% Tris-HCl polyacrylamide gels or 4–15% Tris-HCl polyacrylamide gradient gels (Bio-Rad) in the presence of the reducing agent dithiothreitol (50 μM). Proteins were then transferred to Immobilon paper (Millipore Corp., Bedford, MA) at 110 V for 80 min. After transfer, blots were incubated with primary antibody at 4 C overnight at their respective dilutions: total GR (N499), 1:1,000; rabbit anti-hGR-S203P, 1:10,000; rabbit anti-hGR-S211P, 1:1,000; GR (no. PA3-514, Affinity BioReagents, Golden, CO), 1:500; mouse antihuman -actin (clone AC-15, Sigma, St. Louis, MO), 1:10,000; and mouse antihuman heat-shock protein (HSP) 90 (clone 68, BD Transduction Laboratories, San Jose, CA), 1:1,000. To detect GR, membranes were incubated for 1 h at room temperature with 0.2 μg/ml protein A conjugated to horseradish peroxidase (Kirkegaard & Perry Laboratories, Gaithersburg, MD). To detect -actin and HSP90, the blots were incubated with a 1:5,000 dilution of goat antimouse horseradish peroxidase conjugates. Blots were developed using enhanced chemiluminescence procedures according to the instructions of the manufacturer (Amersham Biosciences, Piscataway, NJ).
For immunoprecipitation studies, 10 μl of anti-GR antiserum (N499) were prebound to protein A/G Plus-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) in PBS at 4 C for 1.5 h and washed with PBS twice to remove the unbound antibody. The antibody-coated beads were incubated overnight at 4 C on a rotator with 1 mg of protein extract from either FIB, CT, or SCT cultures. The beads were then washed five times with PBS and twice with 50 mM Tris (pH 7.5), boiled in 2x sodium dodecyl sulfate sample buffer, and stored at –20 C. Western blotting and immunodetection were carried out as described above using the respective dilutions of total GR and phospho-GR antibodies.
For Western blotting results, intensities of bands on scanned autoradiographs were analyzed using National Institutes of Health Image J software (http://rsb.info.nih.gov/ij). Levels of total GR expression were obtained from normalizing the intensity of the GR band at approximately 97 kDa to that of HSP90 (Fig. 3) or -actin (Fig. 5). For Fig. 5, in which time-dependent down-regulation of GR by DEX treatment was studied, -actin-normalized levels of GR in control and DEX-treated cells were expressed relative to levels at time zero (T0). The effect of DEX treatment on phospho-GR expression (Fig. 3) is expressed as a percentage of control value. Quantitation of all Western blot results was performed for four independent experiments with cells obtained from four different placentas.
Real-time PCR
Real-time PCR was carried out essentially as we have previously described (3). Initially, Tri Reagent (Sigma) was used to extract RNA. Before use in real-time PCR, 2.5 μg of total RNA were reverse-transcribed in a 20 μl reaction containing 50 ng of random hexamers, 0.5 mM of each dNTP, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 5 mM MgCl2, 10 mM dithiothreitol, 40 U of RNase inhibitor, and 50 U of SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). Control reactions were set up without reverse transcriptase to assess the level of contaminating genomic DNA. All reactions were carried out at 25 C for 10 min, 42 C for 50 min, and 70 C for 15 min. The RNA template was removed from the cDNA:RNA hybrid by incubation with 2 U of RNase H at 37 C for 20 min. Amplification was performed on an iCycler iQ real-time PCR detection system (Bio-Rad). Primers for amplification of cDNAs were designed using Beacon Designer software from GenBank cDNA sequences, and the uniqueness of primers was established using BLAST search analysis. The following primers were used: GR (forward, 5'-CCTAAGGACGGTCTGAAGAGC-3'; (478 bp) reverse, 5'-GCCAAGTCTTGGCCCTCTAT-3'); -actin (forward, 5'-TTGGCAATGAGCGGTTCC-3'; (148 bp) reverse, 5'-AGCACTGTGTTGGCGTAC-3'); and 18S rRNA. GR and -actin primers were purchased from Invitrogen. The proprietary 18S rRNA primers from Ambion (QuantumRNA Universal 18S Internal Standard, catalog no. 1718; Austin, TX) were used to amplify this control rRNA sequence. Aliquots containing 25 ng of cDNA were amplified in a total volume of 25 μl containing 12.5 μl of a 2x iQ SYBR Green Supermix and 0.5 μM each primer. All samples were run in triplicate. The reactions were heated at 95 C for 3 min to activate Taq polymerase followed by 40 cycles of 95 C for 10 sec, 56 C for 30 sec (annealing temperature) and 72 C for 30 sec. Melting curves of the products were obtained after cycling by a stepwise increase of temperature from 55–95 C. PCR products were electrophoresed on agarose gels to confirm the presence of a single product of the expected size. Amplified products were sequenced to verify their identity. For the quantification of relative levels of gene expression, the comparative threshold cycle (CT) method was employed as described in User Bulletin No. 2 for ABI PRISM 7700 Sequence Detection System. CT represents the PCR cycle at which an increase in reporter fluorescence above a background signal can first be detected (10 times the SD of the baseline). The 18S rRNA or -actin CT values were subtracted from GR CT values to derive a CT value. The relative expression of the gene of interest was then evaluated using the expression 2–CT. The value for CT was obtained by subtracting the CT of the calibrator (i.e. the 24-h control value within each experimental run) from each CT in that experiment. Cumulative 2–CT results are presented as a mean ± SE obtained in three independent experiments from three different placentas.
Statistics
Results are expressed as a mean ± SE. Data were analyzed by ANOVA or Student’s t test using SigmaStat software from Jandel Scientific (San Rafael, CA). A value of P < 0.05 was considered significant.
Results
Expression of GR in human term placenta
Before analyzing the expression of GR in the placenta, we determined the integrity of the tissue sections by hematoxylin and eosin staining (Fig. 1A). In addition, we established the distribution of cell types within the placental villus by immunohistochemistry using antibodies against cytokeratin 7 to identify cells of trophoblast origin (Fig. 1A, inset), CD45RB to locate immune cells (Fig. 1B), and vimentin to distinguish FIB (Fig. 1C). CT were found as a discontinuous cell layer beneath the SCT (Fig. 1A, inset), whereas FIB were observed throughout the villous core (Fig. 1C). In addition, we observed more than 99% vimentin staining of cultured placental FIB by immunocytochemistry (Fig. 1C, inset). Immune cells, both maternal and fetal, were occasionally observed (Fig. 1B). Having established the cell-type distribution within the placental architecture, we examined GR expression in the human placenta by immunohistochemistry. We observed strong GR immunoreactivity (brown-staining cells) within the nuclei of FIB in the mesenchyme of terminal villi (Fig. 1D). In contrast, CT showed a lower level of GR immunoreactivity in nuclei relative to the FIB, and even lower levels of GR immunoreactivity were noted in nuclei and cytoplasm of SCT (blue-staining cells) (Fig. 1D). Very few macrophages or immune cells were detected in the villous core; the majority of GR-positive cells in the villus were of trophoblast and FIB origin. No staining was observed above background when preimmune serum was employed (Fig. 1D, inset). Thus, the steady-state level of GR differs among FIB, CT, and SCT.
We also examined which cell types exhibit the phosphorylated forms of GR by immunohistochemistry using GR phosphorylation site-specific antibodies to S203 and S211. GR phosphorylation at these sites is indicative of various states of receptor activity. For example, phosphorylation of GR at S211 is strictly agonist-dependent, and positive staining with the GR-S211P antibody is a surrogate marker for this activated form of GR. GR-S203P reactivity was primarily localized to the nuclei of FIB within the villous core, whereas little GR-S203P immunoreactivity was observed in the trophoblast layer (Fig. 1E). The GR-S211P antibody reacted with a majority of the nuclei of placental FIB but with only a minority of nuclei in trophoblasts (Fig. 1F). Again, no staining above background was observed when preimmune sera were employed (Fig. 1, E and F, insets). The results from the immunohistochemical analysis of total GR and phospho-GR expression are summarized in Table 1. In addition to differences in total GR expression, our findings reveal distinct patterns of GR phosphorylation among placental cell types indicative of differences in cell-signaling pathways and GR activation status in vivo. GR appears to be more transcriptionally active in FIB compared with SCT of the term human placenta.
To confirm the cell type-specific pattern of GR expression determined by immunohistochemistry, we cultured primary human placental FIB, CT, and SCT and analyzed GR expression from cell lysates by immunoblotting (Fig. 2A). Note that signal for GR was detected at molecular masses of approximately 97 kDa (denoted by arrow) and 92 kDa in FIB. The 97-kDa band corresponds to the published molecular mass of GR (41), whereas the 92-kDa band was presumed to reflect GR (41). However, Western blotting with an anti-GR antibody did not consistently detect a 92-kDa protein in cell extracts, suggesting that this band is nonspecific. Quantification of GR signals normalized to that of HSP90 revealed greater levels of GR in FIB compared with CT or SCT (Fig. 2B). This result is consistent with our immunohistochemical data demonstrating that patterns of GR expression are faithfully maintained in cultured cells.
We also examined cultured cells for changes in GR phosphorylation as a function of GC treatment (Fig. 3A). In FIB, the GR-S203P antibody recognized GR from both untreated and hormone-treated FIB, and the same pattern was discernible in CT, albeit at a weaker signal intensity. No signal was detected in SCT, most likely due to very low level expression of GR in SCT. The GR-S211P antibody, which showed very weak immunoreactivity toward GR from untreated FIB, displayed substantially more immunoreactivity toward GR from DEX-treated cells. Again, a similar pattern was noted in CT. Based on the relatively high levels of GR expression in FIB, we were able to quantify the effects of DEX treatment on phospho-GR expression in these cells. We noted that DEX treatment significantly enhanced GR-S211 phosphorylation by 2.2-fold compared with control, whereas although the effects on GR-S203 were less pronounced, they also reached statistical significance (Fig. 3B). Thus, GR phosphorylation at S211 is clearly hormone dependent in FIB and is consistent with an important role of phosphorylation and transcriptional activity of GR after GC exposure in this cell type.
We next examined the expression of GR and phospho-GR in the larger stem villi of the placenta by immunohistochemistry to further characterize GR expression in major placental vasculature. FIB were again identified primarily by vimentin staining of the cytoplasm (Fig. 4F), smooth muscle cells were confirmed by expression of -smooth muscle actin (Fig. 4D), and immune cells were located by CD45 staining (Fig. 4E). Immune macrophages were rarely found in the placental mesenchymal core. We found that expression of total GR and GR-S211P were confined to the nuclei of perivascular smooth muscle cells and FIB throughout the core of the villus (Fig. 4, A and C). A low level of GR-S203P staining was noted only in the nuclei of vascular smooth muscle cells (Fig. 4B). Endothelial cells showed low or variable levels of staining for total GR and phospho-GRs relative to smooth muscle cells and FIB. Thus, differentially phosphorylated receptor species are located in specific cell types of the placenta, which likely represent distinct aspects of receptor function in vivo.
Effect of DEX treatment on GR levels in placental FIB
To examine the effect of GC treatment on the level of GR protein in FIB, cells were maintained in medium with or without 100 nM DEX for 7, 24, and 48 h, respectively. Lysates were prepared, and the expression of GR was examined by immunoblotting with an antibody against total GR (Fig. 5A). Levels of GR at T0 and in control and DEX-treated samples were normalized to that of -actin by densitometric analysis of autoradiographs. GR levels in control and DEX-treated cells were then expressed as a percentage of the T0 value. No significant time-dependent change in GR levels for control cells was noted (Fig. 5B). In contrast, DEX treatment reduced GR levels to 27 ± 3, 17 ± 3, and 11 ± 3% of control levels at 7, 24, and 96 h, respectively (Fig. 5A). Thus, GR undergoes rapid hormone-dependent down-regulation in placental FIB.
Effect of DEX treatment on levels of GR mRNA in placental FIB
Previous studies indicated that GC treatment may suppress GR expression through changes in transcription and/or stability of GR protein (31, 33, 34, 42). We used real-time PCR analysis to determine the effects of GC treatment on levels of GR mRNA in FIB. We observed that treatment of FIB with 100 nM DEX for 24 or 48 h did not significantly affect the expression of GR mRNA when normalized to levels of 18S or -actin mRNA (Fig. 6). This suggests that DEX treatment reduced GR expression in FIB through changes in receptor protein stability.
Discussion
GC act via the GR to govern cell differentiation, apoptosis, and response to environmental stresses (43, 44). GR is a transcription factor that is expressed in virtually every mammalian cell, but GC-responsive genes are regulated in a tissue- and cell type-specific manner. Previous studies have demonstrated that GR is expressed in human placenta (19, 20, 21) and that GC treatment alters gene expression in cultures of trophoblasts and FIB isolated from human placentas (2, 3, 7, 38). However, little is known concerning cell type-specific expression of GR and its regulation in human placenta. Our current study clearly demonstrates that FIB in the villous core of the human placenta are targets of GC action. These cells are of critical importance in maintaining placental architecture because ECM proteins synthesized by FIB provide a biological support for CT and SCT layers on the surface of the villus and fetal capillaries below (9, 10, 11, 12). Our initial results using immunohistochemical methods showed that placental FIB express relatively high levels of GR and phospho-GR compared with trophoblast and endothelial cells in terminal villi. Of note was our observation of high levels of GR-S211P in FIB. We previously reported that phosphorylation of GR on S211 was associated with nuclear localization and transcriptional activation of GR after hormone treatment (27). This suggests that placental FIB express transcriptionally active phospho-GR in vivo. In support of this are in vitro studies that showed that DEX treatment enhanced phosphorylation of S211 on the GR of FIB. Of note, Western blotting detected not only the GR species at the predicted molecular mass of 97 kDa (41) (Fig. 2A), but also a 92-kDa protein, the molecular mass reported for GR (41). Our results suggest that the protein detected in our Western blots is most likely not GR because GR antibody did not consistently detect a 92-kDa species (not shown). In addition, the N499 antibody used for immunoprecipitation studies, which recognizes an epitope present in both GR and GR, did not detect the 92-kDa species in these experiments (Fig. 3A).
Our findings from immunohistochemistry and immunoblotting studies using total and phospho-GR antibodies provides insight into how GR is distributed throughout the human placenta and demonstrates how the major placental cell types are differentially phosphorylated. There appears to be a distinct pattern of GR phosphorylation in the placental villus by cell type, which suggests that activation of the GR in each specific cell type may result in selective regulation of cell-specific GR target genes (25). In other words, we suspect that the consistent variation in levels of GR-S203P and GR-S211P in each particular cell type reflects a particular sensitivity to GC treatment and transcriptional activation of specific GR target genes. These results support our previous studies demonstrating GC responsiveness of placental FIB because hormone treatment was found to markedly increase ECM protein expression in FIB isolated from human and baboon placentas (7), whereas other authors have demonstrated that the lung / hydrolase gene is uniquely induced in the lung epithelium A549 cell line (45). Based on our observations that FIB are GC responsive, it is not surprising that the number of doses of antenatal GC given to women at risk for preterm delivery to enhance fetal lung maturation was directly correlated with the severity of villous fibrosis in the placenta (46).
The finding that FIB are targets of GC action is not surprising because GC-regulated genes, including the ECM proteins fibronectin and collagen, are synthesized by placental FIB (3, 12). However, two other GC-regulated genes, CRH and hCG, are synthesized by SCT (4, 5, 47), a cell type shown in the present study to express relatively low levels of GR. This suggests that GC effects in SCT may be indirect or that the low level of GR in SCT is sufficient to promote GC-dependent changes in gene expression. It is of note in the current study that we used a concentration of DEX of 100 nM to elicit GC-dependent responses in placental cells. This concentration of DEX has been previously shown to promote maximal changes in ECM, CRH, and hCG expression in placental and amnion cells by our group and others (3, 4, 5, 7). In addition, 100 nM was the dose of DEX previously used to demonstrate GC-mediated down-regulation of GR (31, 33, 34, 42). The observation that levels of cortisol in maternal sera range from approximately 200–400 nM between 8 wk of pregnancy and term indicates that periplacental levels of GC are high throughout gestation (48).
In the current study, we also report that DEX treatment reduces GR protein expression in FIB between 7 and 48 h of treatment. This phenomenon, known as homologous down-regulation, has been observed in several cell types and has been suggested to represent a mechanism through which cells limit GC responsiveness (31, 32). It is generally thought that homologous down-regulation occurs through cell type-specific mechanisms involving either reduced GR transcription or protein stability and that changes in GR mRNA stability do not play a major role in this process (32). GC-mediated suppression of GR levels has been demonstrated to involve ubiquitination and proteasome-mediated degradation of GR in several cell types (31, 49). Our results suggest that GC-mediated down-regulation of GR levels occurs through changes in protein stability because we observed no change in GR mRNA levels in FIB after DEX treatment.
In conclusion, our studies clearly demonstrate that FIB in the core of the placental villus are a major target of GC action. Further studies will be needed to elucidate how GC and GR interact in the human placenta to determine their role in placental pathophysiology.
Acknowledgments
We are grateful to Dr. Inez Pineda-Torra for critically reading the manuscript.
Footnotes
This work was supported, in part, by National Institutes of Health Grants DK54836 (to M.J.G.) and HD33909 (to S.G.). M.-J.L. is the recipient of a Reproductive Scientist Development Program/Wyeth Award (National Institutes of Health Grant 5K12HD00849).
Abbreviations: CT, Cytotrophoblast(s); CT, threshold cycle; DEX, dexamethasone; ECM, extracellular matrix; FBS, fetal bovine serum; FCS, fetal calf serum; FIB, fibroblast(s); GC, glucocorticoid; GR, glucocorticoid receptor; hCG, human chorionic gonadotropin; hGR, human glucocorticoid receptor; HSP, heat-shock protein; IUGR, intrauterine growth restriction; PBS, phosphate-buffered saline; SCT, syncytiotrophoblast(s); T0, time zero.
References
Guller S, Wozniak R, Krikun G, Burnham JM, Kaplan P, Lockwood CJ 1993 Glucocorticoid suppression of human placental fibronectin expression: implications in uterine-placental adherence. Endocrinology 133:1139–1146
Ryu JS, Majeska RJ, Ma Y, LaChapelle L, Guller S 1999 Steroid regulation of human placental integrins: suppression of 2 integrin expression in cytotrophoblasts by glucocorticoids. Endocrinology 140:3904–3908
Lee MJ, Ma Y, LaChapelle L, Kadner SS, Guller S 2004 Glucocorticoid enhances transforming growth factor- effects on extracellular matrix protein expression in human placental mesenchymal cells. Biol Reprod 70:1246–1252
Ringler GE, Kallen CB, Strauss 3rd JF 1989 Regulation of human trophoblast function by glucocorticoids: dexamethasone promotes increased secretion of chorionic gonadotropin. Endocrinology 124:1625–1631
Jones SA, Brooks AN, Challis JR 1989 Steroids modulate corticotropin-releasing hormone production in human fetal membranes and placenta. J Clin Endocrinol Metab 68:825–830
Ma Y, Ryu JS, Dulay A, Segal M, Guller S 2002 Regulation of plasminogen activator inhibitor (PAI)-1 expression in a human trophoblast cell line by glucocorticoid (GC) and transforming growth factor (TGF)-. Placenta 23:727–734
Ma Y, Lockwood CJ, Bunim AL, Giussani DA, Nathanielsz PW, Guller S 2000 Cell type-specific regulation of fetal fibronectin expression in amnion: conservation of glucocorticoid responsiveness in human and nonhuman primates. Biol Reprod 62:1812–1817
Hahn T, Barth S, Graf R, Engelmann M, Beslagic D, Reul JM, Holsboer F, Dohr G, Desoye G 1999 Placental glucose transporter expression is regulated by glucocorticoids. J Clin Endocrinol Metab 84:1445–1452
Benirschke K, Kaufmann P, eds. 2000 Placental types. In: Pathology of the human placenta. 4th ed. New York: Springer-Verlag; 29–41
Amenta PS, Gay S, Vaheri A, Martinez-Hernandez A 1986 The extracellular matrix is an integrated unit: ultrastructural localization of collagen types I, III, IV, V, VI, fibronectin, and laminin in human term placenta. Coll Relat Res 6:125–152
Yamada T, Isemura M, Yamaguchi Y, Munakata H, Hayashi N, Kyogoku M 1987 Immunohistochemical localization of fibronectin in the human placentas at their different stages of maturation. Histochemistry 86:579–584
Chen CP, Aplin JD 2003 Placental extracellular matrix: gene expression, deposition by placental fibroblasts and the effect of oxygen. Placenta 24:316–325
Benirschke K, Kaufmann P, eds. 2000 Architecture of normal villous trees. In: Pathology of the human placenta. 4th ed. New York: Springer-Verlag; 116–154
Macara L, Kingdom JC, Kaufmann P, Kohnen G, Hair J, More IA, Lyall F, Greer IA 1996 Structural analysis of placental terminal villi from growth-restricted pregnancies with abnormal umbilical artery Doppler waveforms. Placenta 17:37–48
Salafia CM, Minior VK, Pezzullo JC, Popek EJ, Rosenkrantz TS, Vintzileos AM 1995 Intrauterine growth restriction in infants of less than thirty-two weeks’ gestation: associated placental pathologic features. Am J Obstet Gynecol 173:1049–1057
Heckmann M, Wudy SA, Haack D, Pohlandt F 1999 Reference range for serum cortisol in well preterm infants. Arch Dis Child Fetal Neonatal Ed 81:F171–F174
Parker Jr CR, Buchina ES, Barefoot TK 1994 Abnormal adrenal steroidogenesis in growth-retarded newborn infants. Pediatr Res 35:633–636
Seckl JR 1997 Glucocorticoids, feto-placental 11-hydroxysteroid dehydrogenase type 2, and the early life origins of adult disease. Steroids 62:89–94
Driver PM, Kilby MD, Bujalska I, Walker EA, Hewison M, Stewart PM 2001 Expression of 11-hydroxysteroid dehydrogenase isozymes and corticosteroid hormone receptors in primary cultures of human trophoblast and placental bed biopsies. Mol Hum Reprod 7:357–363
Patel FA, Funder JW, Challis JR 2003 Mechanism of cortisol/progesterone antagonism in the regulation of 15-hydroxyprostaglandin dehydrogenase activity and messenger ribonucleic acid levels in human chorion and placental trophoblast cells at term. J Clin Endocrinol Metab 88:2922–2933
Chan CC, Lao TT, Ho PC, Sung EO, Cheung AN 2003 The effect of mifepristone on the expression of steroid hormone receptors in human decidua and placenta: a randomized placebo-controlled double-blind study. J Clin Endocrinol Metab 88:5846–5850
Yamamoto KR 1985 Steroid receptor regulated transcription of specific genes and gene networks. Annu Rev Genet 19:209–252
Hollenberg SM, Weinberger C, Ong ES, Rosenfeld MG, Evans RM 1985 Primary structure and expression of a functional human glucocorticoid receptor cDNA. Nature 318:635–641
Bamberger CM, Bamberger AM, de Castro M, Chrousos GP 1995 Glucocorticoid receptor , a potential endogenous inhibitor of glucocorticoid action in humans. J Clin Invest 95:2435–2441
Ismaili N, Garabedian MJ 2004 Modulation of glucocorticoid receptor function via phosphorylation. Ann NY Acad Sci 1024:86–101
Krstic MD, Rogatsky I, Yamamoto KR, Garabedian MJ 1997 Mitogen-activated and cyclin-dependent protein kinases selectively and differentially modulate transcriptional enhancement by the glucocorticoid receptor. Mol Cell Biol 17:3947–3954
Wang Z, Frederick J, Garabedian MJ 2002 Deciphering the phosphorylation "code" of the glucocorticoid receptor in vivo. J Biol Chem 277:26573–26580
Cidlowski JA, Cidlowski NB 1981 Regulation of glucocorticoid receptors by glucocorticoids in cultured HeLa S3 cells. Endocrinology 109:1975–1982
Svec F, Rudis M 1981 Glucocorticoids regulate the glucocorticoid receptor in the AtT-20 cell. J Biol Chem 256:5984–5987
Lacroix A, Bonnard GD, Lippman ME 1984 Modulation of glucocorticoid receptors by mitogenic stimuli, glucocorticoids and retinoids in normal human cultured T cells. J Steroid Biochem 21:73–80
Wallace AD, Cidlowski JA 2001 Proteasome-mediated glucocorticoid receptor degradation restricts transcriptional signaling by glucocorticoids. J Biol Chem 276:42714–42721
Schaaf MJ, Cidlowski JA 2002 Molecular mechanisms of glucocorticoid action and resistance. J Steroid Biochem Mol Biol 83:37–48
Dong Y, Poellinger L, Gustafsson JA, Okret S 1988 Regulation of glucocorticoid receptor expression: evidence for transcriptional and posttranslational mechanisms. Mol Endocrinol 2:1256–1264
McIntyre WR, Samuels HH 1985 Triamcinolone acetonide regulates glucocorticoid-receptor levels by decreasing the half-life of the activated nuclear-receptor form. J Biol Chem 260:418–427
Rogatsky I, Zarember KA, Yamamoto KR 2001 Factor recruitment and TIF2/GRIP1 corepressor activity at a collagenase-3 response element that mediates regulation by phorbol esters and hormones. EMBO J 20:6071–6083
Tassone P, Tuccillo F, Bonelli P, D’Armiento FP, Bond HM, Palmieri C, Tagliaferri P, Venuta S 2002 Fetal ontogeny and tumor expression of the early thymic antigen UN1. Int J Oncol 20:707–711
Fant ME, Nanu L 1995 Human placental endothelin: expression of endothelin-1 mRNA by human placental fibroblasts in culture. Mol Cell Endocrinol 109:119–123
Guller S, LaCroix NC, Kirkun G, Wozniak R, Markiewicz L, Wang EY, Kaplan P, Lockwood CJ 1993 Steroid regulation of oncofetal fibronectin expression in human cytotrophoblasts. J Steroid Biochem Mol Biol 46:1–10
Kliman HJ, Nestler JE, Sermasi E, Sanger JM, Strauss 3rd JF 1986 Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 118:1567–1582
Douglas GC, King BF 1989 Isolation of pure villous cytotrophoblast from term human placenta using immunomagnetic microspheres. J Immunol Methods 119:259–268
Gupta S, Gyomorey S, Lye SJ, Gibb W, Challis JR 2003 Effect of labor on glucocorticoid receptor (GR(Total), GR, and GR) proteins in ovine intrauterine tissues. J Soc Gynecol Investig 10:136–144
Rosewicz S, McDonald AR, Maddux BA, Goldfine ID, Miesfeld RL, Logsdon CD 1988 Mechanism of glucocorticoid receptor down-regulation by glucocorticoids. J Biol Chem 263:2581–2584
Munck A, Mendel DB, Smith LI, Orti E 1990 Glucocorticoid receptors and actions. Am Rev Respir Dis 141:S2–S10
Sapolsky RM, Romero LM, Munck AU 2000 How do glucocorticoids influence stress responses Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr Rev 21:55–89
Rogatsky I, Wang JC, Derynck MK, Nonaka DF, Khodabakhsh DB, Haqq CM, Darimont BD, Garabedian MJ, Yamamoto KR. 2003 Target-specific utilization of transcriptional regulatory surfaces by the glucocorticoid receptor. Proc Natl Acad Sci USA 100:13845–13850
Ghidini, A, Pezzullo JC, Sylvestre G, Lembet A, Salafia CM 2001 Antenatal corticosteroids and placental histology in preterm birth. Placenta 22:412–417
Riley SC, Walton JC, Herlick JM, Challis JR 1991 The localization of corticotropin-releasing hormone in the human placenta and fetal membranes throughout gestation. J Clin Endocrinol Metab 72:1001–1007
Dorr HG, Heller A, Versmold HT, Sippell WG, Herrmann M, Bidlingmaier F, Knorr D 1989 Longitudinal study of progestins, mineralocorticoids, and glucocorticoids throughout human pregnancy. J Clin Endocrinol Metab 68:863–868
Sengupta S, Wasylyk B 2001 Ligand-dependent interaction of the glucocorticoid receptor with p53 enhances their degradation by Hdm2. Genes Dev 15:2367–2380(Men-Jean Lee, Zhen Wang, )