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Endocrinology, doi:10.1210/en.2006-1212
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Endocrinology Vol. 148, No. 8 3645-3654
Copyright © 2007 by The Endocrine Society

Corticotropin-Releasing Hormone Receptor Type 1 and Type 2 Mediate Differential Effects on 15-Hydroxy Prostaglandin Dehydrogenase Expression in Cultured Human Chorion Trophoblasts

Lu Gao, Ping He, Jinyan Sha, Chunmin Liu, Ling Dai, Ning Hui and Xin Ni

Departments of Physiology (L.G., P.H., X.N.) and Gynecology and Obstetrics (J.S., C.L., L.D., N.H.), Second Military Medical University, Shanghai 200433, People’s Republic of China

Address all correspondence and requests for reprints to: Dr. Xin Ni, Department of Physiology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, People’s Republic of China. E-mail: nxljq2003{at}yahoo.com.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Throughout gestation, the chorion laeve controls the levels of biologically active prostaglandins (PGs) by its high level of nicotinamide adenine dinucleotide-dependent 15-hydroxy PG dehydrogenase (PGDH). In this study, we investigate the effects mediated by CRH receptors on the expression of PGDH in the chorion. We found that both CRHR1 and CRHR2 were localized in cultured chorion trophoblast cells, with CRH-R1{alpha}, R1ß, R1c, R1e, and R1f and CRHR2ß isoforms identified in these cells. To block the actions of endogenous CRH and its related peptides, cultured chorion trophoblasts were treated with an increasing concentration of {alpha}-helical CRH 9–41, the nonselective CRH receptor antagonist, which resulted in decreased mRNA and protein expression as well as the activity of PGDH. To investigate the individual role of CRHR1 and CRHR2, cell cultures were treated with the specific CRHR1 antagonist antalarmin and CRHR2 antagonist astressin2B, respectively. The results showed that antalarmin increased whereas astressin2B decreased mRNA and protein expression as well as the activity of PGDH in chorion cells. When the cells were treated with an exclusive CRHR2 agonist, urocortin II, elevated expression and activity of PGDH was exhibited. However, cells treated with either exogenous CRH or urocortin I showed significantly increased PGDH expression, and these effects could be blocked by astressin2B but not by antalarmin. We suggest that, in chorion trophoblast cells, CRHR1 and CRHR2 mediate divergent effects on PGDH expression, and this may provide a precise regulation of PGs levels from chorion to myometrium during pregnancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROSTAGLANDINS (PGs) PRODUCED in intrauterine tissue play a central role in the initiation and progression of labor in most mammalian species (1, 2). Specifically, it has been shown that PGs induce uterine contractility and play a role in regulating changes in extracellular matrix metabolism associated with ripening of the cervix (3, 4). In addition, PGs have also been shown to play an important role in up-regulation of the fetal hypothalamic-pituitary-adrenal axis, membrane rupture, and maintenance of uterine and placental blood flow (5, 6). PGs are formed from arachadonic acid, released from membrane stores, by the action of PGH synthase (7). The initial oxidative step of metabolism of PGs uses the enzyme nicotinamide adenine dinucleotide-dependent 15-hydroxy-PG dehydrogenase (PGDH) to catalyze the reversible oxidation of the E and F series PGs to form biologically inactive 15-keto metabolites (8). Throughout gestation, the amnion membrane produces large amounts of PGE2, whereas the chorion laeve, situated between the amnion and the decidua, not only produces PGE2 and PGF2{alpha}, but also controls the myometrial concentration of active PGs due to its very high PGDH activity (9, 10, 11). These intensive metabolic influences suggest that the changing of PG metabolic rates in the chorion may substantially alter bioactive PG levels in the uterus. Previous studies have shown that levels of mRNA encoding PGDH and PGDH activity were lower in chorion from patients at term spontaneous labor, compared with term elective cesarean section, and these levels decreased further in chorion collected from patients at idiopathic preterm labor and preterm labor with underlying infection (12, 13). These observations underscore the critical role of chorionic PGDH in the biological mechanisms that control parturition. However, our understanding of the regulation of PGDH in chorion is still incomplete.

In the past decade, CRH has been implicated in playing a key role in the control of human pregnancy and parturition (14). It has been demonstrated that the biosynthesis and secretion of placental CRH increases exponentially with advancing gestation and this increase is mirrored by exponential increases in CRH concentration in maternal plasma (14, 15, 16). Abnormally elevated maternal CRH levels during pregnancy are associated with preterm delivery, whereas unusually low levels correlate with prolonged gestations (14, 17). Throughout gestation, CRH is synthesized not only by the placenta, but also by fetal membranes including chorion (18, 19). Although the function of CRH in the fetal membrane is unknown, it has been shown that increasing levels of CRH were expressed in fetal membranes, including the chorion trophoblasts, at term and preterm (20). Recently, other members of CRH family including urocortin I (UCNI), urocortin II (UCNII), and urocortin III (UCNIII) have also been shown to be synthesized by fetal membrane throughout gestation (21, 22, 23).

All members of the CRH family exert their effect by binding to CRH receptors. Two major CRH receptor subtypes, CRHR1 and CRHR2, have been identified that belong to the class II G protein-coupled receptor superfamily. These receptors share 70% identity at the amino acid level, but have different binding properties for the members of the CRH family (24, 25, 26). UCNI has been reported to bind to both CRHR1 and CRHR2, but with higher affinity for CRHR2. CRH has approximately 10-fold lower affinity than UCNI for CRHR2, whereas UCNII and UCNIII bind exclusively to CRHR2. The human fetal membranes have been shown to express mRNA encoding both subtypes of CRH receptor, suggesting that this tissue is a target for CRH and its related peptides during pregnancy (19, 27, 28). More recently, McKeown and Challis (29) showed that CRH stimulates PGDH activity in cultured chorion trophoblasts in a paracrine/autocrine fashion. However, a detailed and quantitative analysis of the role of CRH in the regulation of PGDH at the protein and mRNA levels, as well as the specific CRH receptor subtype that is responsible for this CRH activity has not been performed.

In the present study, we conducted experiments to determine the protein expression of CRH receptor subtypes in chorion cells and determine quantitatively the role of CRH receptor subtypes in the actions of CRH and its related peptides on the expression as well as the activity of PGDH in the primary chorion cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chorionic trophoblast cell culture
Thirty one human fetal membranes were obtained from elective cesarean section patients with singleton pregnancies at term without labor. Tissue collections were performed with approval of Changhai hospital human ethics committees and informed consent was received from patients. Chorionic trophoblasts were isolated and cultured according to the modified method of Kliman et al. as described previously (30, 31). Briefly, About 40 g of chorion were peeled off the amnion and then minced. The minced chorionic tissue was dispersed with 0.125% trypsin (Invitrogen Corp., Carlsbad, CA) and 0.02% collagenase (Worthington, Lakewood, NJ) in DMEM (Sigma Chemical Co., St. Louis, MO) containing 0.1% BSA, three times for 60 min each time. The cells were then loaded onto a discontinuous Percoll (Amersham Biosciences, Uppsala, Sweden) gradient (5–70%), then centrifuged at 2500 x g for 20 min. Cytotrophoblast cells between the density of 1.049 and 1.062 g/ml were collected and plated in six-well plates (Corning, Inc. Costar Corp., Cambridge, MA) at a density of 3 x 106 cells per well for determining mRNA and protein expression of PGDH, or in 24-well plates (Corning, Inc. Costar Corp.) at a density of 1 x 106 cells per well for determining PGDH activity. Then cells were cultured in phenol red-free DMEM containing 10% fetal calf serum (FCS) at 37 C in 5% CO2-95% air.

On the third day after plating, the cells were changed to FCS-free DMEM containing one of the following treatments: {alpha}-helical CRH 9–41 (0–10–5 M) (Calbiochem, La Jolla, CA), a nonselective antagonist of CRH receptors; antalarmin (0–10–6 M) (Sigma Chemical Co.), the selective antagonist to CRHR1; or astressin2B (0–10–6 M), the selective antagonist to CRHR2 (Sigma Chemical Co.); CRH (0–10–6 M) (Sigma Chemical Co.); UCNI (0–10–6 M) (Sigma Chemical Co.); or UCNII (0–10–6 M) (Calbiochem) in the presence or absence of antalarmin (10–6 M) and astressin2B (10–6 M). Control cultures were maintained without additives, and each treatment was performed in triplicate for each preparation of cells for 24 h.

At the end of each experiment, representative wells of cells were fixed and immunostained for cytokeratin and vimentin using primary antibodies (Dako, Inc., Carpinteria, CA) at a dilution 1:1000 to assess cell purity.

Immunofluorescence analysis
Trophoblast cells were grown for 3 d and then fixed in 4% paraformaldehyde for 1 h after washing with PBS. Fixed cells were washed with PBS and incubated with 10% BSA for 1 h. Then the cells were incubated with antibodies raised against human CRHR1 (sc-12381; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or human CRHR2 (sc-20550; Santa Cruz Biotechnology, Inc.) at a dilution 1:500 or human 15-OH PGDH (Cayman Chemical, Ann Arbor, MI) at a dilution of 1:1000. The CRHR1 antibody is directed against an epitope between amino acid positions 81 and 109 of CRHR1 of human origin, where no sequence homology exists to CRHR2, and all CRHR1 subtypes with the exception of CRHR1g are detected. The antibody against CRHR2 was raised against a peptide mapping near the C terminus of CRHR2 of human origin and recognizes all of CRHR2 isoforms.

All dilutions were made in 1% BSA in PBS. This incubation was performed overnight at 4 C. Subsequently, the specimens were washed with PBS three times and then incubated with CY3-conjugated antimouse IgG (1:100) for cytokeratin or CY3-conjugated antirabbit IgG (1:100) for PGDH and fluorescein isothiocyanate-conjugated antigoat IgG (1:100) for CRHR1 or CRHR2 at 37 C for 1 h in the dark. For negative controls, the primary antibody was either substituted with a normal IgG in same dilution or preabsorbed with 5:1 (wt/wt) of peptide/antibody. Results were viewed under fluorescent microscope using appropriate filters.

Total RNA extraction and RT-PCR
After 24-h treatment of cells with various agents, the cells were mechanically dispersed by scraping with a rubber policeman for 1 min in the presence of TRIzol (Invitrogen, Grand Island, NY) reagent and then incubated them for 5 min at the room temperature to permit complete dissociation of nucleoprotein complex. Total RNA was prepared from individual samples by using TRIzol reagent according to the manufacturer’s guidelines. The purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis before use. Two-microgram RNA was reverse transcribed with oligo(dT)18 primer using the Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) and then stored at –20 C. Specific primers for the amplification of the CRHR2 variants and CRHR1{alpha}/CRHR1ß were used as described previously (32) (Table 1Go). PCR solution consisted of 2.0 µl diluted cDNA, 0.4 µM of each paired primers, 2.5 mM Mg2+, 250 µM deoxynucleotide triphosphates, 2 U Taq DNA polymerase (Promega), and 1x PCR buffer. PCR was set at 94 C (45 sec), 58 C (45 sec), 72 C (1 min) in a total of 40–80 cycles with a final extension step at 72 C for 10 min.


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  		TABLE 1. List of primers used for the amplification of CRHR2 and CRHR1{alpha}/ß isoforms in human chorion trophoblast cells

 
Primers for nested PCR were previously described (33), and were designed to distinguish the different variants of CRHR1 (Table 2Go). RT products (cDNA) from cells were used as template for the first round of PCR. After 40 cycles of amplification, 2 µl of the reaction mixture was used for additional 40-cycle amplification. The primers used in this second round of amplification were internal to the first set of primers.


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  		TABLE 2. List of nested PCR primers used for the amplification of CRHR1 isoforms in human chorion trophoblast cells

 
Ten microliters of the reaction mixture was subsequently electrophoresed on a 1.5% agarose gel and visualized by ethidium bromide, using a 100-bp DNA ladder (Invitrogen) to estimate the band sizes. As a negative control for all of the reactions, distilled water was used in place of cDNA. The identity of the PCR products was confirmed by sequencing. Sequence data were analyzed using Blast nucleic acid database searches from the National Centre for Biotechnology Information.

Quantitative real-time RT-PCR
To measure PGDH mRNA levels in response to CRH, CRH-related peptide, and CRH receptor antagonist treatments, quantitative real-time PCR was carried out using Rotor-Gene 3000 (Corbett Research, Sydney, Australia). The nucleotide sequences of the primers for PGDH were: sense 5'-TTGCACAGCAGCCGGTTTAT-3', antisense 5'-TTGGCAATCAATGGTGGGTC-3' (accession number NM_000860). The reaction solution consisted of 2.0 µl diluted cDNA, 0.2 µM of each paired primer, 200 µM deoxynucleotide triphosphates, 2 U Taq DNA polymerase (Promega), and 1x PCR buffer. SYBR Green (BMA, Rockland, ME) was used as detection dye. Quantitative real-time PCR conditions were optimized according to preliminary experiments to achieve linear relationships between initial RNA concentration and PCR product. The annealing temperature was set at 61 C, and amplification cycles were set at 40 cycles. The temperature range to detect the melting temperature of the PCR product was set from 60–95 C. Amplification of the housekeeping gene ß-actin (accession number NM_001101) was measured for each sample as an internal PCR control for sample loading and normalization. The specificity of the primers was verified by examining the melting curve as well as subsequent sequencing of the real-time RT-PCR products. The sizes of amplicons for PGDH and ß-actin were 247 and 275 bp, respectively. To determine the relative quantitation of gene expression for both target and housekeeping genes, the comparative threshold cycle (Ct) method with arithmetic formulae was used (34). Subtracting the Ct of housekeeping gene from the Ct of the target gene yields the {Delta}Ct in each group (control and experimental groups), which was entered into the equation 2–{Delta}Ct and calculated for the exponential amplification of PCR. PGDH mRNA levels were normalized relative to ß-actin values.

Western blotting analysis
Cells were scraped off the plate in the presence of lysis buffer consisting of 60 mM Tris-HCl, 2% SDS, 10% sucrose, 2 mM phenylmethylsulfonyl fluoride (Merck, Darmstadt, Germany), 1 mM sodium orthovanadate (Sigma Chemical Co.), and 10 µg/ml aprotinin (Bayer, Leverkusen, Germany). The cell lysates were quickly sonicated and centrifuged at 12,000 x g for 5 min at 4 C. The supernatant was collected and protein concentration was assayed using a modified Bradford assay. The samples were diluted in sample buffer [250 mM Tris-HCl (pH 6.8), containing 4% SDS, 10% glycerol, 2% ß-mercaptoethanol, and 0.002% bromophenol blue] and boiled for a further 5 min. Aliquots of proteins were separated by SDS-PAGE (10%) and subsequently transferred to nitrocellulose membranes by electroblotting. The membrane was blocked in 5% skim milk powder in 0.1% Tris-buffered saline/Tween 20 at room temperature for 2 h, and then was incubated with antibody raised against human PGDH (Cayman Chemical) at a dilution 1:1000 overnight at 4 C. After three washes with Tris-buffered saline/Tween 20, the membrane was incubated with a secondary horseradish peroxidase-conjugated antibody for 1 h at room temperature. Immunoreactive proteins were visualized using the enhanced chemiluminescence Western blotting detection system (Santa Cruz Biotechnology, Inc.). The light-emitting bands were detected with x-ray film. The resulting band intensities were quantitated using an image scanning densitometer (Furi Technology, Shanghai, China). To control sampling errors, the ratio of band intensities for PGDH to ß-actin was obtained to quantify the relative protein expression level.

PGDH activity assay
PGDH activity was assayed by measuring 13,14-dihydro-15-keto PGF2{alpha} (PGFM), the stable metabolite of PGF2{alpha}. PGFM content in culture medium was measured by using a commercial PGFM enzyme immunoassay kit (Cayman Chemical) according to the manufacturer’s protocol. The standard curve ranged from 1000 to approximately 7.81 pg/ml. The sensitivity of the assay was 8.2 pg/ml, and the intraassay coefficients of variation were less than 10.6%.

The conditions of PGDH activity assay including concentration of PGF2{alpha}, the substrate of PGDH, and incubation time were optimized at the cell density of 1 x 106 cells per well (in 24-well plates). After the cells were treated with increased concentration of phorbol myristate acetate (PMA), the medium was replaced with FCS-free DMEM containing various concentrations of PGF2{alpha} (10, 50, 80, 100 ng/ml), and then incubated for another 1–8 h. The media were collected and stored at –80 C for later assay. The results showed that the maximum reaction was obtained in the presence of 80 ng/ml PGF2{alpha} and at the incubation time of 4 h in the cells either treated or nontreated with PMA (10–6 M). The increased activity of PGDH activity was achieved in the cells treated with increased concentration of PMA (10–8–10–6 M) for 24 h. Therefore, the following experiments were performed in the conditions of the substrate concentration at 100 ng/ml and the incubation time at 4 h. The levels of PGFM in supernatants of the cells without addition of PGF2{alpha} and in the media with addition of PGF2{alpha} in the absence of cells were undetectable.

RIA of CRH and UCNI
After chorion cells were cultured for 72 h, the culture media were replaced with fresh DMEM without FCS and then incubated for another 3–6 h. Culture media were collected and stored at –80 C for later assay.

CRH and UCNI RIA were performed as previously described (35). Synthetic Tyr-CRH or Tyr-UCNI was iodinated by the chloramine T method, purified by Sephadex G-25 gel chromatography, and used as a tracer. UCNI antibody and CRH antibody were supplied by Phoenix Biotech Co. Ltd (Beijing, China). UCNI assay did not cross-react with rat/human CRH, somataostatin, neuropeptide Y, and LHRH. CRH assay did not cross-react with ACTH, sauvagine, LHRH, and prepro-CRH. The sensitivity of the assay for CRH and UCNI was 3.2 and 6.4 pg/ml, respectively.

Statistics
For illustrative purposes, the results are presented as the mean percent control ± SEM. Control cultures were conducted in the absence of exogenous CRH, UCNI, UCNIII, antalarmin, or astressin2B. One-way ANOVA followed by the Student-Newman-Keuls test was used to assess significant differences between each two groups. Significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell morphology
At the end of the culture period, chorion trophoblasts were present as clumps of cells or as single cells. Immunostaining of the cells with cytokeratin or vimentin showed that more than 90% of the cells were positive for cytokeratin and less than 1% of the cells were positive for vimentin (data not shown).

Expression of CRHR1 and CRHR2 in cultured chorion trophoblasts
Eight isoforms of CRHR1 have been described (33, 36, 37), which were derived from different intron/extron splicing as shown in Fig. 1AGo. We used the specific primers for CRHR1{alpha}/CRHR1ß and a nested RT-PCR protocol to screen for the presence of these variants in cultured chorion trophoblasts. Cultured chorion trophoblasts were found to express CRHR1{alpha}, CRHR1ß, CRHR1c, CRHR1e, and CRHR1f isoforms (Fig. 1Go, B and C). We were unable to detect other CRHR1 variants in cultured chorion cells.


Figure 1
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  		FIG. 1. Schematic representation and PCR analysis of the human CRHR1 isoforms. A, CRHR1 has eight different receptor isoforms generated by alternative exon splicing. B, Primers are indicated by arrows above A that go across exons 2–7 and 9–14. Isoforms are distinguished by different molecular weight of amplified bands. Bands indicated by arrow 1 can be {alpha}, d, f, or g isoforms. Bands indicated with arrow 2 are specific for isoform c. Arrow 3 indicates CRHR1e. Bands indicated with arrow 4 can only be {alpha}, ß, c, e. Arrow 5 indicates CRHR1f. This schematic of receptor splice variants is based on Ref. 33 . C, The existence of CRHR1ß was confirmed by another set of primers as described in Materials and Methods.

 
Three isoforms of CRHR2, termed CRHR2{alpha}, CRHR2ß, and CRHR2{gamma}, were found previously (38, 39, 40). PCR amplification using specific primers for the CRHR2 isoforms resulted in the detection only of CRHR2ß in chorion trophoblasts (Fig. 2CGo). None of the other CRHR2 isoforms (CRHR2{alpha} and CRHR2{gamma}) were detected. To confirm that the negative outcome for CRHR2{alpha} and CRHR2{gamma} was due to a lack of expression although not the failure of the primers to work effectively in the PCR systems, we used cDNA generated from human myometrium and brain tissue biopsies (both from Changhai Hospital, all consents informed) as positive control for CRHR2{alpha} and CRHR2{gamma}, respectively. In addition, up to 80 cycles of PCR were performed. The results suggest that the unique set of primers work effectively in the PCR system and the negative outcome is due to a lack of expression (Fig. 2Go, B and D).


Figure 2
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  		FIG. 2. PCR analysis of the human CRHR2 isoforms. A, CRHR2 has three different receptor isoforms. Specific primers for each isoform are indicated by arrows that go across the exons. B, CRHR2{alpha}; C, CRH2ß; and D, CRHR2{gamma}. 1, Chorion cells; 2, positive control (human myometrium for CRH2{alpha} and human brain tissue biopsies for CRHR2{gamma}); 3, negative control.

 
To investigate the CRH receptor protein expression in the chorion trophoblast cultures, immunofluorescence was carried out using CRHR1- or CRHR2-specific antibody. A strong positive staining for CRHR1 and CRHR2 was revealed in plasma membrane and cytoplasm of these cells (Fig. 3Go, A and B). To confirm the type of trophoblasts that were immunostained for the receptor, double staining was performed on the same specimens for CRHR1 and cytokeratin or CRHR2 and cytokeratin. We found that cells expressing CRHR1 or CRHR2 were also positive for cytokeratin (Fig. 3Go, C–F).


Figure 3
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  		FIG. 3. Immunofluorescence analysis of CRH receptors in cultured chorion trophoblast cells. Immunostained with antibody against CRHR1 (A) and CRHR2 (B). C and D, The same cells were immunostained for cytokeratin that was used to confirm the type of trophoblast cells. CRHR1 and cytokeratin (E) or CRHR2 and cytokeratin (F) overlie. Negative controls, the primary antibody was either substituted with normal goat IgG (G) or normal mouse IgG (H). A–H, Original magnifications, x400.

 
Immunofluorescent staining confirmed the presence of PGDH in chorion trophoblast cultures. Double staining for CRHR1 and PGDH or CRHR2 and PGDH showed that most of cells that expressed PGDH were also positive for CRHR1 or CRHR2 (Fig. 4Go).


Figure 4
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  		FIG. 4. Double immunostaining for CRH receptors and PGDH in cultured chorion trophoblasts. Immunostaining for CRHR1 (A) and CRHR2 (B) in cells. C and D, The same cells were immunostained for PGDH. CRHR1 and PGDH (E) or CRHR2 and PGDH (F) overlie. Negative control (preabsorption) for CRHR1 (G) or CRHR2 (H). Negative control, primary antibody was replaced with normal rabbit IgG (I) or primary antibody was preabsorbed with PGDH blocking peptide (J). A–J, Original magnifications, x400.

 
Effects of CRH receptor antagonists on PGDH expression
It is known that human chorion trophoblasts express CRH, UCNI, UCNII, and UCNIII. We measured CRH and UCNI content in culture media of chorion cells, and found that CRH content was 117 ± 46 pg/ml up to 3 h incubation, whereas UCNI content was 32 ± 6.7 pg/ml up to 6 h incubation. To block the effects of these endogenous CRHRs agonists, we used the CRHR1/R2 nonselective antagonist {alpha}-helical CRH 9–41. As shown in Fig. 5Go, A and B, {alpha}-helical CRH 9–41, at 10–6 and 10–5 M, resulted in a significant decrease in both mRNA and protein levels of PGDH after a 24-h treatment period. To determine the individual role of two CRH receptor subtypes on PGDH expression, cells were treated with the CRHR1-specific antagonist antalarmin and the CRHR2-specific antagonist astressin2B, respectively. Treatment of chorion cell cultures with increasing concentration of antalarmin increased the levels of PGDH mRNA dose dependently at the concentrations of 10–8–10–6 M (Fig. 5CGo). Cells that were treated with astressin2B showed a significant decrease in PGDH mRNA expression in a dose-dependent manner (Fig. 5EGo). Western blot analysis also showed that antalarmin increased whereas astressin2B decreased PGDH protein expression (Fig. 5DGo).


Figure 5
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  		FIG. 5. Effects of CRHR antagonists on transcript and protein levels of PGDH in cultured chorion trophoblast cells. Real-time RT-PCR and Western blot analysis were used to quantify PGDH mRNA and protein levels, respectively. A and B, Effects of {alpha}-helical CRH 9–41 on mRNA and protein expression of PGDH. C and D, Changes in mRNA expression of PGDH induced by antalarmin and astressin2B. E, Effects of antalarmin (10–6 M) and astressin2B (10–6 M) on PGDH protein expression. Ctr, Control; {alpha}-CRH, {alpha}-helical CRH 9–41; Anta, antalarmin; Astr, astressin2B. Values are presented as mean percent control ± SEM for five cultures from five patients (n = 5) performed in triplicate. *, P < 0.05; **, P < 0.01 compared with control.

 
Effects of exogenous CRH, UCNI, and UCNII on PGDH expression
To further determine the effects of CRH and the urocortins on PGDH expression, we treated chorion cells with CRH, UCNI, and UCNII, respectively. CRH significantly increased both mRNA and protein levels of PGDH at the concentrations of 10–8–10–6 M. Astressin2B could block CRH-stimulated PDGH mRNA and protein expression, whereas antalarmin was not able to block the actions of CRH. (Fig. 6Go, A and B). UCNI exhibited similar effects as CRH, increasing both PGDH mRNA and protein expression at concentrations of 10–9–10–6 M. The effects of UCNI on PGDH expression could be reversed by astressin2B (Fig. 6Go, C and D).


Figure 6
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  		FIG. 6. Effects of CRH and its related peptides on PGDH expression in cultured chorion trophoblast cells. A and B, Cells were treated for 24 h with CRH (10–10–10–6 M) in the presence or absence of antalarmin (10–6 M) and astressin2B (10–6 M). mRNA and protein levels of PGDH were assessed by real-time RT-PCR and Western blot analysis, respectively. C and D, Treatment of cells with UCNI (10–9–10–6 M) in the presence or absence of antalarmin (10–6 M) and astressin2B (10–6 M); E and F, effects of UCNII (10–9–10–6 M) in the presence or absence of astressin2B (10–6 M). Values are presented as mean percent control ± SEM for five cultures from five patients (n = 5) performed in triplicate. Ctr, Control; Anta, antalarmin; Astr, astressin2B. *, P < 0.05; **, P < 0.01 compared with control; #, P < 0.05 compared with CRH 10–7 mol/liter (A and B); #, P < 0.05 compared with UCNI 10–7 mol/liter (C and D); #, P < 0.05 compared with UCNII 10–7 mol/liter (E and F).

 
As expected, treatment of cells with UCNII at the concentration of 10–9–10–6 M resulted in significantly increased PGDH mRNA and protein expression. Maximum effect was obtained at the 10–6 M, which caused a 2.5-fold increase in both PGDH mRNA and protein expression. Addition of astressin2B reversed the increase in PGDH caused by the addition of UCNII (Fig. 6Go, E and F).

Effects of CRH receptor antagonists and CRH and its related peptides on PGDH activity
To confirm that CRH receptor antagonists, CRH, and urocortins also influence PGDH activity, PGDH activity in chorion cells was assayed after 24-h treatments of cells with the peptides and CRH receptor antagonists. As shown in Fig. 7AGo, {alpha}-helical CRH 9–41 (10–6 M) significantly decreased PGDH activity. Astressin2B also decreased PGDH activity, but antalarmin increased PGDH activity. Cells were treated with exogenous CRH, UCNI, and UCNII, which resulted in increased PGDH activity. These effects were blocked by astressin2B (Fig. 7Go, B–D).


Figure 7
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  		FIG. 7. Effects of CRH receptor antagonists, CRH and its related peptides on PGDH activity. PGDH activity was assayed by measuring PGFM concentration in cell cultures with an enzyme immunoassay kit. A, Cells were treated with {alpha}-helical CRH 9–41 (10–6 M), antalarmin (10–6 M), or astressin2B (10–6 M) for 24 h. B and C, Treatment of cells with CRH (10–6 M), UCNI (10–6 M) in the presence or absence of antalarmin (10–6 M) or astressin2B (10–6 M). D, Effects of UCNII (10–7 M) in the presence or absence of astressin2B (10–6 M). Ctr, Control; {alpha}-CRH, {alpha}-helical CRH 9–41; Anta, antalarmin; Astr, astressin2B. Values are presented as mean percent control ± SEM for five cultures from five patients (n = 5) performed in triplicate. **, P < 0.01 compared with control. #, P < 0.05 compared with CRH 10–7 M (B); #, P < 0.05 compared with UCNI 10–7 M (C); #, P < 0.05 compared with UCNII 10–7 M (D).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have demonstrated that most of the cells that were labeled with PGDH also expressed CRHR1 or CRHR2, confirming that these cells are targets of CRH and its related peptides. Our study and others have demonstrated that chorion trophoblasts are capable of secreting CRH and UCNI in vitro (18, 21). More recently, Imperatore et al. (23) demonstrated that human chorion cytotrophoblasts express UCNII and UCNIII peptides, suggesting that chorion trophoblasts are the source of endogenous CRHR1 and CRHR2 ligands during pregnancy. The present study showed that when CRHRs were blocked by {alpha}-helical CRH 9–41, a CRHR1/R2 antagonist, there is a significant decrease in PGDH mRNA and protein expression. To investigate the role of CRHR1 and CRHR2 in the modulation of PGDH expression, we treated cultured chorion trophoblasts with selective CRHR1 and CRHR2 antagonists, respectively. We showed that CRHR1 antagonist resulted in a significant increase in PGDH mRNA and protein expression. However, when CRHR2 activity was blocked in the cells by the CRHR2 antagonist astressin2B, mRNA and protein expression of PGDH was significantly decreased. These data indicate that endogenous CRH and its related peptides, produced locally in chorion trophoblasts, enhance PGDH expression by activation of CRHR2 but inhibit PGDH expression through CRHR1.

The studies of McKeown and Challis (29) suggested that locally produced CRH stimulates PGDH activity by demonstrating that the nonselective CRH receptor antagonist astressin as well as specific CRH antibody decreased PGDH activity in cultured chorion trophoblasts. In this study, we found that another nonselective CRH receptor antagonist, {alpha}-helical CRH 9–41, reduced not only PGDH activity, but PGDH mRNA and protein expression in cultured chorion trophoblasts. These suggest that enhancement of PGDH activity by endogenous CRH and its related peptides might be due to their stimulatory effects on PGDH expression. In addition, treatment of cells with the CRHR1/R2 agonists, CRH and UCNI, modulated PGDH expression and activity preferentially via the CRHR2 subtype, as shown by the use of specific antagonists. Taken together, this leads us to suggest that, in the model of cultured chorion trophoblasts, up-regulation of PGDH expression and activity mediated by CRHR2 is predominant, and CRHR1 and CRHR2 exhibited opposite actions on PGDH expression. However, in vivo, which subtype of CRHRs that predominates in regulating chorionic PGDH expression requires further investigation.

Both CRHR1 and CRHR2 have multiple known mRNA splice variants. Currently, eight variants of the mRNA for CRHR1, termed R1{alpha}-h, have been described (33, 36, 37), and these should encode different sized proteins. However, the functions and exact expression levels of these isoforms have been difficult to determine due to the lack of available antibodies to the splice variants. Several lines of evidence have demonstrated that CRHR1{alpha} is the main functional CRHR1 variant (32, 41, 42, 43). Other CRHR1 variants have distinctive binding and signaling properties. For instance, CRHR1c has decreased binding capacity, whereas CRHR1e appears to attenuate CRHR1{alpha} signaling in coexpression experiments (44, 45). The CRHR2 gene has three mRNA splice variants, encoding R2{alpha}, R2ß, and R2{gamma} receptor isoforms (38, 39, 40). The CRHR2ß is about 10-fold more potent in second messenger activation compared with CRHR2{alpha} or R2{gamma} although their agonist binding and signaling properties of the various CRH-related peptides are not significantly different (40). In this study, we not only identified CRHR1{alpha}, the functional CRHR1 variant, but also CRHR1ß, CRHR1c, CRHR1e, and CRHR1f variants in cultured chorion trophoblasts, suggesting that activation of CRHR1{alpha} by endogenous CRH and its related peptides might be influenced by the existence of other variants of CRHR1 in chorion cells. With regard for CRHR2, only CRHR2ß, the most potent CRHR2 variant, was detected in these cells. This expression pattern of the CRH receptor variants in cultured chorion trophoblasts may partly explain that the modulation of PGDH is predominantly mediated by CRHR2 in these cells.

Bioactive PGs have been implicated as integral uterotonins in the labor process, and two important enzymes, PGH synthase 2 and PGDH, control their synthesis and metabolism, respectively. PGDH located within the chorion has been postulated to play a crucial role in maintaining a metabolic barrier to bioactive PGs, controlling the levels of bioactive PGs entering the decidua and myometrium (46, 47), where they could cause early uterine activation. In addition to expressing PGDH, the human chorion secretes CRH and other CRH-related peptides throughout gestation, and these peptides can bind to the CRHRs that are expressed in chorion trophoblasts (18, 21, 23). The results from the present study suggest that CRH, and its related peptides, may act in an autocrine/paracrine fashion to play an important role in the local regulation of PGDH expression throughout gestation. With increasing gestational age, CRH levels in placental and fetal membrane increase, whereas UCNI levels in placenta are unchanged (20, 48, 49). However, levels of both CRH and UCNI in maternal plasma are elevated during labor (49, 50). Although the levels of the CRH-related peptides UCNII and UCNIII are unknown during pregnancy, we speculate that this rise of CRH and UCNI may result in decreased PGDH expression through an interaction with CRHR1 and CRHR2, and contribute locally to the appropriate transfer of bioactive PGs to the myometrium. Differential regulation of PGDH by CRHR1 and CRHR2 in chorion may provide a precise means of regulating the PG levels entering the decidua and myometrium during pregnancy.


   Acknowledgments
 
We thank the nursing and medical staff of the delivery suites at Changhai Hospital for their cooperation in obtaining placenta, and Dr. Richard C. Nicholson (John Hunter Hospital, Newcastle, Australia) for his helpful suggestion in the preparation of the manuscript.


   Footnotes
 
This work was supported by Natural Science Foundation of China (No. 30170982) and Program for Changjiang Scholars and Innovative Research Team in University (No. IRT0528).

Disclosure Summary: All authors have nothing to disclose.

First Published Online April 26, 2007

Abbreviations: Ct, Threshold cycle; FCS, fetal calf serum; PG, prostaglandin; PGDH, 15-hydroxy PG dehydrogenase; PGFM, 13,14-dihydro-15-keto PGF2{alpha}; PMA, phorbol myristate acetate; UCNI, II, and III, urocortins I, II, and III.

Received September 5, 2006.

Accepted for publication April 17, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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L. Gao, C. Lu, C. Xu, Y. Tao, B. Cong, and X. Ni
Differential Regulation of Prostaglandin Production Mediated by Corticotropin-Releasing Hormone Receptor Type 1 and Type 2 in Cultured Human Placental Trophoblasts
Endocrinology, June 1, 2008; 149(6): 2866 - 2876.
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