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Original Studies |
Departments of Obstetrics and Gynecology (Y.M., G.K., C.J.L., L.L., S.G.) and Biochemistry (S.G.), New York University Medical Center, New York, New York 10016
Address all correspondence and requests for reprints to: Dr. Seth Guller, Department of Obstetrics and Gynecology, New York University Medical Center, Tisch Hospital Room 531, 550 First Avenue, New York, New York 10016.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the etiology of labor is poorly understood, it is clear that parturition is associated with a marked increase in the concentration of glucocorticoid (GC) and CRH in amniotic fluid and maternal and fetal plasma (8, 9, 10). Recent data demonstrated that plasma levels of biologically active CRH were elevated in women who delivered preterm and were lower in those women who delivered postterm (11). As the placenta is the major source of plasma CRH during pregnancy (12), these findings are consistent with the hypothesis that a "placental clock" controls the length of gestation (11). The finding that GCs increase CRH expression in the placenta and fetal membranes (13) suggests an important role of GC in regulating gestational length.
Previous data obtained in our laboratory demonstrated that GCs specifically and coordinately suppressed the synthesis of extracellular matrix (ECM) proteins in cytotrophoblasts isolated from human placentas (6, 7, 14, 15). We hypothesize that the parturition-dependent increase in GC may down-regulate ECM protein expression at regions of uterine-placental contact and may be required for separation of the placenta after expulsion of the fetus. The purpose of the present study was to establish cell type-specific patterns and mechanism(s) of GC-mediated suppression of FN expression in human placental cells as a model to elucidate the physiological regulation of ECM protein production at the uterine-placental interface. Using primary cell cultures isolated from term placentas, we report that the chronically expressed, matrix-suppressive action of GC in human placenta is specific to cytotrophoblasts and is mediated through a short lived, cycloheximide (CHX)-sensitive protein intermediate. Elucidation of the mechanism of GC effects on ECM protein expression in the human placenta is of immediate clinical relevance in light of the current recommendation for the antenatal use of GC for enhancement of fetal lung maturity (16).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Culture media and calf serum were obtained from Flow Laboratories (McLean, VA). FCS was purchased from HyClone Laboratories (Logan, UT). Collagenase was purchased from Worthington Biochemical Corp. (Lakewood, NJ). Laboratory plasticware was purchased from Falcon, Becton Dickinson Labware (Lincoln Park, NJ). ITS+, a mixture containing insulin, transferrin, and selenium, was obtained from Collaborative Research-Becton Dickinson (Bedford, MA). Dexamethasone (DEX), CHX, actinomycin D, and monoclonal antibody to human plasma FN (clone FN-15) were obtained from Sigma Chemical Co. (St. Louis, MO). Ultraspec used to isolate ribonucleic acid (RNA) was purchased from Biotecx Laboratories (Houston, TX). [32P]Deoxy-CTP, [32P]UTP, and [35S]Protein Labeling Mix were purchased from New England Nuclear (Boston, MA). Deoxyribonuclease (DNase), ribonuclease (RNase) inhibitor, and protease K were obtained from Boehringer Mannheim (Indianapolis, IN). Plasmids containing complementary DNAs (cDNAs) to human FN and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from the American Type Culture Collection (Rockville, MD). Zeta-Probe nylon membranes were purchased from Bio-Rad (Richmond, CA).
Methods
Cell culture. Tissues were obtained from pregnancies with normally grown, singleton fetus. Pregnancies were not complicated by preterm labor, preterm rupture of membranes, or other pathological conditions. The study protocol received approval from the institutional review board committee at New York University Medical Center. Placentas (n = 15) were transported to the laboratory immediately after delivery by cesarean section and were trimmed free of connective tissue and decidua. Cytotrophoblasts were isolated by methodologies developed in our laboratory (6, 7) based on published protocols (17, 18). Cytotrophoblasts were isolated from villous tissue after trypsin digestion and centrifugation on a continuous Percoll gradient (6, 7).
Human placental fibroblasts were isolated based on procedures described by Fant and Nanu (19). Briefly, washed villi were digested for 45 min in a 1:1 mixture of phenol red-free Ham’s F-12-DMEM (basal medium) containing 0.1% collagenase and 0.01% DNase. Dispersed cells were filtered through a 160-µm pore size stainless steel sieve and seeded in T-75 culture flasks in basal medium supplemented with 10% charcoal-stripped calf serum and ITS+ (i.e. SCS medium). At confluence, the cells were passaged, and second to fourth passage cells were used for experiments. Cytotrophoblasts and fibroblasts were maintained at 37 C in a humidified atmosphere of 5% CO2-95% air in SCS medium.
Protein labeling and immunoprecipitation
Levels of protein synthesis in cytotrophoblasts treated with and without DEX and CHX were determined as we have previously described (6). Briefly, cells were incubated for 4 h in methionine-free SCS medium containing 15 µCi/mL [35S]Protein Labeling Mix, and levels of cell-associated trichloroacetic acid (TCA)-precipitable radioactivity were determined (6). For immunoprecipitation experiments, cells were labeled for 4 h with 75 µCi/mL [35S]Protein Labeling Mix. Approximately 106 TCA-precipitable counts per min of labeled medium were incubated with a 1:50 dilution of anti-FN antibody and protein G-Sepharose (7). Washing of immune complexes and SDS-PAGE were performed as we previously described (7).
Northern blotting and FN messenger RNA (mRNA) stability
RNA was extracted from cytotrophoblasts using the Ultraspec protocol, a modification of the method of Chomczynski and Sacchi (20). Approximately 15–25 µg total RNA (based on A260) were separated on a 1% agarose gel containing 2.2 mol/L formaldehyde (14). After transfer of RNA to Zeta-Probe nylon membranes, levels of FN and GAPDH mRNAs were detected using 32P-labeled cDNA probes as previously described (14). For FN mRNA stability studies, cells were incubated with and without 10-7 mol/L DEX for 48 h. Fresh medium with and without 10-7 mol/L DEX containing 1 µg/mL actinomycin D was then added for the indicated time. RNA was extracted from cells, and Northern blotting was performed. The zero time point reflects cells harvested before the addition of medium supplemented with actinomycin D.
Run-on transcription assay
Cytotrophoblasts (2 x 107 cells/experimental
point) maintained 48 h with and without 10-7 mol/L
DEX were washed with ice-cold phosphate-buffered saline, scraped, and
lysed on ice in buffer containing 0.6% Nonidet P-40, 0.15 mol/L NaCl,
10 mmol/L Tris (pH 7.9), 1 mmol/L ethylenediamine tetraacetate (EDTA),
0.2 mmol/L phenylmethylsulfonylfluoride (PMSF), and 0.5 mmol/L
dithiothreitol (DTT), i.e. lysis buffer. The lysed cells
were centrifuged (5 min, 2500 rpm) at 4 C, and the supernatant was
discarded. The nuclear pellet was washed in cold lysis buffer and
resuspended in 100 µL of a solution containing 75 mmol/L NaCl, 0.5
mmol/L EDTA, 50% glycerol, 1 mmol/L DTT, 1 mmol/L PMSF, and 20 mmol/L
Tris, pH 8.1. The integrity of the nuclei was verified microscopically
after staining with methylene blue or propidium iodide. Nuclei were
stored at -80 C until use. For assay, thawed nuclei were resuspended
in 100 µL transcription buffer containing 25 mmol/L NaCl; 0.3 mmol/L
EDTA; 0.7 mol/L (NH4)SO4; 8 mmol/L
MnCl2; 20 mmol/L creatine phosphate; 200 µg/mL creatine
kinase; 2.1 mg/mL heparin; 0.1 mmol/L PMSF; 1.5 mmol/L DTT; 600 U RNase
inhibitor; 1 mmol/L each of ATP, CTP, and GTP; 300 µCi
[
-32P]UTP; and 180 mmol/L Tris, pH 8.1. After a 20-min
incubation at 26 C, 2 µL transfer RNA (40 mg/mL), 20 µL
MgCl2 (0.1 mol/L), and 10 µL DNase (10,000 U/mL) were
added, and the mixture was incubated for 20 min at 26 C with vortexing
every 5 min. The mixture was then treated with 2 µL protease K (20
mg/mL), 24 µL SDS (10%), 3 µL EDTA (0.5 mol/L), and 30 µL Tris
(200 mmol/L), pH 7.4, and incubated at 37 C for 30 min. The newly
transcribed RNA was isolated after extraction with
phenol-chloroform-isoamyl alcohol (25:24:1), chloroform alone, and
precipitation with ethanol in the presence of 0.3 mol/L sodium acetate
(pH 5.2). The pellet was washed once in 80% ethanol and resuspended in
80 µL of a solution of 154 mmol/L NaCl, 1 mmol/L EDTA, and 10 mmol/L
Tris, pH 7.4. Labeled RNA was separated from unincorporated labeled UTP
by passage through a NucTrap push column (Stratagene, La Jolla, CA).
Approximately 107 cpm labeled RNA were then hybridized to
Zeta-Probe membrane containing 5 µg FN, GAPDH plasmids, and a plasmid
devoid of insert (i.e. pBR322 negative control) according to
instructions provided by the manufacturer (Bio-Rad, Hercules, CA).
Immunoassay for onfFN
Levels of onfFN in culture media were determined by an immunoassay using FDC-6 monoclonal antibody as we previously described (6, 7). The concentration of cellular protein was quantitated using the DC Protein Assay from Bio-Rad Laboratories (Hercules, CA).
Data analysis
Quantitation of autoradiographic signals and printing of data were carried out using Sigma Scan/Sigma software (St. Louis, MO) as we previously described (21). Levels of FN expression are expressed as the mean ± SEM. Statistical analysis was carried out using Student’s t test (SigmaStat software, Jandel, San Rafael, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For experiments, cytotrophoblasts and fibroblasts isolated from
human term placentas were maintained in SCS medium with and without
10-7 mol/L DEX, and levels of FN mRNA and protein were
determined using Northern blotting, immunoprecipitation, and
enzyme-linked immunosorbent assay (ELISA) procedures. We observed that
DEX treatment down-regulated levels of FN mRNA and protein in
cytotrophoblasts to 13–19% of control levels (Fig. 1
and Table 1).
Conversely, GC treatment increased FN expression in fibroblasts to
164–310% of control levels, as measured in Northern blotting and
ELISA assays (Table 1). This suggests that GC-mediated suppression
of FN expression is specific to cytotrophoblasts.
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mRNA stability assays (n = 4) were conducted to elucidate a
mechanism of GC-mediated down-regulation of FN expression in
cytotrophoblasts. Cells treated for 48 h with and without
10-7 mol/L DEX were then maintained for the indicated time
with the transcription inhibitor actinomycin D (1 µg/mL), and the
decay in FN mRNA over time was examined by Northern blotting. The
experiment shown in Fig. 2 revealed that
DEX treatment did not markedly alter levels of FN mRNA compared to
control values, suggesting that the suppressive action of GC on FN
expression in cytotrophoblasts was not mediated through changes in
message stability.
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Run-on assays using nuclei isolated from cytotrophoblasts were
performed to examine the effects of GC treatment on transcription of
the FN gene. Levels of FN and GAPDH transcription were 2- to 3-fold
greater than those in the negative transcription control condition in
which labeled RNA was hybridized to plasmid devoid of FN insert
(indicated by PBR in Fig. 3). This
suggested that cytotrophoblasts expressed relatively low rates of
transcription. In four independent experiments, FN gene transcription
in DEX-treated cells was 96.7 ± 2.2% of control levels when
normalized to levels of GAPDH transcription, suggesting that GC
treatment may not markedly affect rates of FN gene transcription in
cytotrophoblasts.
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Studies using CHX, an inhibitor of protein synthesis, were
performed to determine whether the GC-mediated suppression of FN
expression in cytotrophoblasts was mediated through a protein
intermediate. Cells were treated for 48 h with and without
10-7 mol/L DEX and CHX at the indicated concentration, and
levels of FN mRNA were normalized to levels of GAPDH mRNA after
Northern blotting. In the absence of CHX, DEX treatment reduced FN mRNA
levels to 18.6% of control values (Fig. 4). In contrast, in the presence of 125,
250, and 500 ng/mL CHX, the levels of FN mRNA in DEX-treated cells were
115.9%, 106.0%, and 90.0% of control values (i.e. cells
maintained with CHX in the absence of DEX), respectively. Lower
concentrations of CHX (1–10 ng/mL) were also effective in reversing
the GC-mediated inhibition of FN expression (not shown). In five
independent experiments in which cells were maintained with and without
100–125 ng/mL CHX and 10-7 mol/L DEX for 48 h, the
presence of CHX increased levels of FN mRNA in DEX-treated cells from
18.2 ± 4.4% to 90.8 ± 6.2% of control levels. These
values were statistically different at the P < 0.0001
level. A time course comparing the effect of DEX treatment on levels of
FN mRNA in the presence and absence of CHX is presented in Fig. 5. In the absence of CHX treatment, DEX
promoted a reduction in levels of FN mRNA to 6.4% and 7.1% of control
levels on days 2 and 4 of culture, respectively. In contrast, in the
presence of CHX, the levels of FN mRNA in DEX-treated cells were
86.6%, 98.1%, 88.8%, and 35.3% of control levels on days 1, 2, 3,
and 4 of culture, respectively. These results indicated that CHX
treatment rapidly reversed the DEX-mediated inhibition of FN expression
(i.e. GC treatment no longer down-regulated levels of FN
mRNA compared to control values) and suggest that a protein
intermediate is involved in GC-mediated suppression of FN expression in
cytotrophoblasts.
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Cytotrophoblasts were maintained for 48 h with and without
10-7 mol/L DEX and the indicated concentration of CHX
(Fig. 6), and the rate of protein
synthesis, expressed as TCA-precipitable radioactivity, was examined
after metabolic labeling of cells with 35S-labeled amino
acids. Of note, CHX treatment significantly (P <
0.0001) reduced the rate of protein synthesis in control and
DEX-treated cells only when used at concentrations of 1000 and 3000
ng/mL. This indicated that the DEX-mediated suppression of FN mRNA
expression was reversed at a concentration of CHX (i.e.
100–125 ng/mL) that did not affect the overall level of protein
synthesis. In addition, we observed that in the presence and absence of
CHX, the rate of protein synthesis was unaffected by DEX treatment
(Fig. 6), suggesting that the effects of DEX in cytotrophoblasts were
not due to a coordinate reduction in protein synthesis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study we observed that GC suppressed the synthesis of FN mRNA and protein in cytotrophoblasts isolated from human term placentas to approximately 15% of control levels. We also noted that the effects of DEX on FN expression were not mediated through changes in FN mRNA stability. In addition, we observed that GC treatment had little effect on the transcription of the FN gene in cytotrophoblasts as measured in run-on assays. We cannot rule out the possibility that GC may affect FN transcription in cytotrophoblasts, as the levels of transcription in nuclei isolated from control cells were only 2- to 3-fold above background levels. To date, to the best of our knowledge, there are no reported studies of hormone- or growth factor-mediated modulation of promoter/reporter gene activity in transfected primary cultures of cytotrophoblasts isolated from human term placentas. It was recently observed that only extremely small recombinant adenovirus-based systems were effective in transducing cytotrophoblasts and only before their syncytialization in culture (25).
In the current report we carried out experiments with CHX, an inhibitor of protein synthesis, to determine whether the effects of GC on FN expression were direct or required de novo synthesis of protein. We observed that the presence of 100 ng/mL CHX reversed the GC-mediated suppression of FN mRNA. Our experiments also showed that the effects of CHX were rapid (i.e. they occurred within the first 24 h of treatment) and were observed at concentrations at which the overall level of protein synthesis in cytotrophoblasts was not affected. These results are consistent with the conclusion that suppression of FN expression by GC in cytotrophoblasts requires de novo protein synthesis and is mediated through the action of a short lived intermediate, the synthesis of which is inhibited at low concentrations of CHX.
GC suppression of cytokine gene expression (i.e. its
antiinflammatory action) may not be a direct action of the hormone,
as it was reversed in the presence of low concentrations of CHX (26).
Recent data suggested that antiinflammatory effects of GCs may be
mediated in part through the action of a cytoplasmic inhibitor protein,
I
B (26, 27). The results obtained in the present study are
consistent with an important role of an intermediate, perhaps an
IB-like protein, in GC-mediated suppression of FN expression in
cytotrophoblasts. However, it must be noted that GC effects in
cytotrophoblasts were more chronic in nature (
48 h) than the acute
actions of GC (1–4 h) in the reports (26, 27) discussed above.
There is a well documented elevation in levels of GC in amniotic fluid and maternal and fetal sera in association with parturition whether occurring before or at term (8, 28). Labor was associated with a reduction in collagen expression in the separation zone of the placenta from the placental bed (29). Although cytotrophoblast-derived onfFN is present at high concentrations in the zone of placental separation (4, 5) and Nitabuch’s fibrin (30), it has not been determined whether labor affects the distribution and/or expression of onfFN at these sites. Our results suggest that a GC-induced protein intermediate may inhibit the synthesis of onfFN by cytotrophoblasts, reduce uterine-placental adherence, and be associated with separation of the placenta from the uterus after expulsion of the fetus. Therefore, it is of prime importance to identify mediators of the matrix-suppressive action of GCs in human placental cytotrophoblasts.
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Acknowledgments |
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Footnotes |
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Received March 17, 1997.
Revised October 2, 1997.
Accepted October 31, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R, Lockwood CJ, Ma Y, DiPasquale B, Guller
S. 1996 Expression of Fas ligand by human cytotrophoblasts:
implications in placentation and fetal survival. J Clin Endocrinol
Metab. 81:3119–3122.B in
mediation of immunosuppression by glucocorticoids. Science. 270:283–286.B activity through induction of IB synthesis. Science. 270:286–290.This article has been cited by other articles:
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  J. P. van Beek, H. Guan, L. Julan, and K. Yang Glucocorticoids Stimulate the Expression of 11{beta}-Hydroxysteroid Dehydrogenase Type 2 in Cultured Human Placental Trophoblast Cells J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5614 - 5621. [Abstract] [Full Text] [PDF]
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 M.-J. Lee, Y. Ma, L. LaChapelle, S. S. Kadner, and S. Guller Glucocorticoid Enhances Transforming Growth Factor-{beta} Effects on Extracellular Matrix Protein Expression in Human Placental Mesenchymal Cells Biol Reprod, May 1, 2004; 70(5): 1246 - 1252. [Abstract] [Full Text] [PDF]
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Y. Ma, C. J. Lockwood, A. L. Bunim, D. A. Giussani, P. W. Nathanielsz, and S. Guller Cell Type-Specific Regulation of Fetal Fibronectin Expression in Amnion: Conservation of Glucocorticoid Responsiveness in Human and Nonhuman Primates Biol Reprod, June 1, 2000; 62(6): 1812 - 1817. [Abstract] [Full Text]
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 J. S. Ryu, R. J. Majeska, Y. Ma, L. LaChapelle, and S. Guller Steroid Regulation of Human Placental Integrins: Suppression of {alpha}2 Integrin Expression in Cytotrophoblasts by Glucocorticoids Endocrinology, September 1, 1999; 140(9): 3904 - 3908. [Abstract] [Full Text]
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 L. I. McKay and J. A. Cidlowski Molecular Control of Immune/Inflammatory Responses: Interactions Between Nuclear Factor-{kappa}B and Steroid Receptor-Signaling Pathways Endocr. Rev., August 1, 1999; 20(4): 435 - 459. [Abstract] [Full Text] [PDF]
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