help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bartsch, O.
Right arrow Articles by Ivell, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bartsch, O.
Right arrow Articles by Ivell, R.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 1 324-334
Copyright © 2004 by The Endocrine Society

Phosphodiesterase 4 Inhibition Synergizes with Relaxin Signaling to Promote Decidualization of Human Endometrial Stromal Cells

Olaf Bartsch, Bettina Bartlick and Richard Ivell

Institute for Hormone and Fertility Research, University of Hamburg, 20251 Hamburg, Germany

Address all correspondence and requests for reprints to: Dr. Olaf Bartsch, Institute for Hormone and Fertility Research, University of Hamburg, Falkenried 88, 20251 Hamburg, Germany. E-mail: bartsch{at}ihf.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The decidualization of endometrial stromal cells in the secretory phase of the menstrual cycle is an essential prerequisite for the implantation of a blastocyst. This profound differentiation process is accompanied by sustained elevated intracellular cAMP concentrations in vivo. Primary cell cultures of endometrial stromal cells decidualize by treatment with cAMP-elevating agents in vitro. Our previous findings indicated that the cAMP-degrading activities of phosphodiesterases (PDE) and signaling of the peptide hormone relaxin are coupled in human endometrial stromal cells. In the present study we have chosen a pharmacological approach to test whether relaxin binding and PDE inhibition cooperate to induce decidualization. Measurement of PDE activity and relaxin-stimulated cAMP accumulation in the presence of diverse PDE inhibitors identified PDE4 and PDE8 as the principal PDE isoforms involved in human endometrial stromal cells. The PDE4 inhibitor rolipram was most effective in elevating intracellular cAMP concentrations and synergizing with relaxin to achieve maximal in vitro decidualization, as determined by measurement of the expression of the decidual marker genes for prolactin and IGF-binding protein-1 and measurement of prolactin secretion. Gene expression for PDE4D and PDE4C was significantly up-regulated during in vitro decidualization. Treatment of cell cultures with the protein kinase A inhibitor H89 revealed a minor role for protein kinase A-mediated positive feedback control of PDE4 activity in human endometrial stromal cells, consistent with sustained elevated cAMP essential for decidualization in vitro. These findings introduce the new idea of clinically applying the combination of a specific PDE4 inhibitor with an effector such as relaxin, thereby offering an alternative nonsteroidal luteal phase support for the endometrium to encourage endometrial development and implantation in subfertile women undergoing assisted reproductive technology procedures.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING THE MENSTRUAL cycle, the stromal cells of the endometrium undergo a progressive and controlled differentiation process to meet the evolutionary demands of providing an adequate maternal interface for the implanting blastocyst. Before ovulation, the endometrium is in a proliferative phase, with few relatively underdeveloped glands and spiral arteries. This phase is typified by stromal cells of a relatively undifferentiated mesenchymal phenotype. After ovulation, when the concentration of circulating progesterone increases, the stromal cells of the proliferative endometrium differentiate, changing shape when cultured to a more polygonal phenotype. This process of stromal cell decidualization is accompanied by a marked development of both endometrial glands and spiral arteries. Adequate decidualization is essential for the endometrium to be able to support implantation of a newly formed embryo. At a molecular level, decidualization is characterized by the up-regulation of genes such as those for prolactin (PRL) (1), IGF-binding protein-1 (IGF-BP1) (2), or glycodelin (3, 4), which are among the major secretory proteins of the human decidualized endometrium during pregnancy. Additional factors are secreted in vivo, which are necessary for correct epithelial differentiation, such that the latter cells can properly accommodate an implanting blastocyst in the so-called window of implantation (5, 6, 7).

Two of three blastocysts fail to implant in otherwise healthy women. In subfertile and infertile women, this proportion is considerably higher. Although part of this implantation failure can be attributed to poor embryo quality, there may also be a marked endometrial deficit in the capacity of the endometrium to support implantation. Endometrial development is known to be important for in vitro fertilization outcome (8, 9, 10), and supplementary hormonal (luteal) support has been recommended to improve the receptivity of the endometrium (11, 12). Furthermore, disruption of endometrial differentiation is associated with several gynecological disorders, such as endometriosis, adenomyosis, and luteal phase defect, and is an important cause of infertility and menstrual disorders (13, 14, 15).

Although the importance in vivo of the steroid hormones estradiol and progesterone is unquestioned, their precise mechanism of action in the endometrium is not known. At a cell biological level, decidualization of endometrial stromal cells can be induced in vitro by a high concentration of progesterone after estradiol priming (16) as well as by a variety of agents that share in common the ability to increase the intracellular concentration of cAMP. These include natural hormones such as relaxin (16, 17), prostaglandin E2, or gonadotropins, such as human chorionic gonadotropin or LH (18), as well as reagents like 8-bromo-cAMP (19) or the adenylate cyclase activator forskolin (19).

Intracellular cAMP concentrations are the result of an interplay between cAMP generation and hydrolysis by specific phosphodiesterases (PDE). The latter provide a powerful means of manipulating the magnitude and duration of the biological response to cAMP. We and others have shown previously that the ability of hormones such as relaxin to induce the production of cAMP can be enhanced by the addition of general inhibitors of PDE activity, such as 3-isobutyl-1-methylxanthine (IBMX) (20). Indeed, recent experiments have suggested that relaxin may even be signaling through a regulation of endogenous PDE activity (20, 21).

The present study was undertaken to investigate the possible role of PDEs in the regulation of intracellular cAMP in human endometrial stromal cells, particularly in combination with the peptide hormone relaxin, and hence to determine whether PDE inhibitors may be useful agents with which to promote decidualization and thereby to support implantation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and drug treatments

All patient samples were collected in accord with the Helsinki Declaration and the authorization of the local ethical committee. Human endometrial stromal cells (ESC) were prepared from uterine samples of premenopausal and cycling woman (35–50 yr old) who were undergoing hysterectomy for leiomyoma. Routinely, primary cultures of more than 95% purity were obtained and cultured using established methods (20, 22). Each experiment used tissue from a single individual. Human ESC were cultured in T80 flasks (Nunc, Roskilde, Denmark) in basal medium containing DMEM-Ham’s F-12 at a 1:1 ratio, 10% fetal calf serum that had been depleted of steroids by treatment with dextran-coated charcoal, 100 IU/ml penicillin, 100 µg/ml streptomycin, 1 µg/ml insulin, and 10-9 M 17ß-estradiol (Sigma-Aldrich Corp., Deisenhofen, Germany). Cells were taken between passages 2 and 6 and were finally transferred to 6- and 12-well plates (Nunc) or to 92X17 TC dishes (Nunc), as indicated. Progesterone (Sigma-Aldrich Corp.) and/or relaxin (>85% pure porcine relaxin, giving a single Coomassie-stained band in PAGE; courtesy of Dr. O. D. Sherwood) were added to the medium where indicated at concentrations of 250 nM and 100 ng/ml, respectively. Previous studies had shown the EC50 for this porcine relaxin in ESC to be about 1.4 nM (equivalent to 10 ng/ml porcine relaxin) (20).

Acute effects of PDE inhibition on the intracellular cAMP concentration after a 20-min exposure to relaxin were measured using a cAMP fluorometric immunoassay (FIA; see below). Confluent cultures of ESC in 12-well plates were preincubated for 30 min with the PDE-selective inhibitors rolipram, Ro-20-1724, and zaprinast (Calbiochem, La Jolla, CA) at concentrations ranging from 1–100 µM or with the nonselective inhibitors IBMX (Calbiochem) and papaverine (Sigma-Aldrich Corp.) at concentrations ranging from 1 µM to 1 mM. Additionally, ESC cultures were pretreated with 10 µM of the protein kinase A (PKA) inhibitor H89 (Calbiochem) for 60 min before incubation with PDE inhibitors and/or relaxin.

Longer-term studies of the effect of PDE inhibition on the in vitro decidualization of ESC were performed in six-well plates by coincubating relaxin and/or progesterone with 100-µM final concentrations of either rolipram or papaverine using incubation times from 48–144 h, as indicated. Alternatively, 8-bromo-cAMP (8Br-cAMP; 1 mM: BioLog, Bremen, Germany) was added to the medium as a positive control. Medium was changed every 48 h. Cells were harvested for RNA preparation (see below), or conditioned medium was collected and stored at -80 C until measurement of secreted PRL (see below). All PDE inhibitors were dissolved in dimethylsulfoxide (DMSO) and supplemented to cultures to give a final concentration of 0.1% DMSO, which was used as a negative control in all experiments. The human monocyte cell-line THP-1 was purchased from the European Cell Culture Collection (no. 88081201, Porton Down, UK) and cultured as described previously (20).

cAMP determination

ESC in 12-well plates were incubated with combinations of relaxin and different inhibitors of PDE as described above. For measurement of intracellular cAMP, confluent monolayers were washed twice with ice-cold PBS and extracted in 1 ml cold ethanol at -20 C overnight. The cAMP content was measured using a conventional ELISA as described previously (23) or a modification of this assay using a time-resolved FIA, as follows. After centrifugation of any cell debris, cell lysates in 70% ethanol were evaporated to dryness, taken up in Eagle’s PBS buffer [E-PBS; 0.1 M sodium phosphate (pH 7.0), 0.15 M NaCl, 5 mM EDTA, 0.2% BSA, and 0.01% thimerosal], and acetylated with 0.05% (vol/vol, final concentration) of freshly prepared acetylation mixture (1 vol acetic anhydride plus 2 vol triethylamine). Acetylated cAMP was then measured by FIA, with a europium (Eu3+) chelate-labeled cAMP derivative (cAMP-diethylenetriamine-pentaacetic acid, from Sigma-Aldrich Corp.)-EU3+ as tracer, competing with the cAMP in the sample for binding to a cAMP-specific rabbit polyclonal antibody. For detection, Eu3+ was dissociated from the tracer by incubation with a commercially available enhancement solution (Wallac 1244-105, PerkinElmer Life Sciences, Boston, MA) rapidly to form a new, highly fluorescent complex. Finally, samples were pulsed with excitation light of 340 nm, and emitted light was detected at 615 nm in a Wallac Victor2 1420 multilabel counter (PerkinElmer Life Sciences). In detail, microtiter strips (FluoroNunc module 437915, coated with goat antirabbit {gamma}-globulin) were removed from the freezer and equilibrated to room temperature. Three hundred microliters of E-PBS buffer per well were incubated for 1 min at room temperature and decanted. Subsequently, 50 µl (50,000 counts/sec) of sample/well were pipetted first, followed by 50 µl tracer solution [cAMP-diethylenetriamine-pentaacetic acid-EU3+ dissolved in 0.1 M Tris-HCl (pH 7.5), 0.1% NaN3, 0.9% NaCl, 0.01% Tween 20, and 0.1% BSA] second and 100 µl antibody [affinity-purified antiserum (MS1 rabbit) diluted 1:300 in E-PBS buffer containing 0.0005% (wt/vol) methyl orange] third. After incubation at 4 C for 20 h in the dark, assay plates were washed four times with 300 µl cold wash solution (0.02% Tween 20 and 0.5% NaCl)/well. Empty wells were then filled with 200 µl enhancement solution and incubated for 60 min at room temperature on a shaker in a dust-free chamber before measuring time-resolved fluorescence. The FIA permitted measurements in the range from 0.04–9.72 pmol/ml (corresponding to 2–486 fmol/well). Within this range, the intra- and interassay coefficients of variants of measured samples were generally less than 15%. The slope factor of the calibration curve was -1.07, and the EC50 was 0.32 pmol cAMP/ml. The specificity of the assay was determined by measuring cross-reactivity with structurally analogous compounds (e.g. cGMP, 2'3'-cAMP, and other common nucleotides). Cross-reactivity for all tested compounds was less than 0.0003%.

Human PRL (hPRL) ELISA

ESC in six-well plates were incubated with combinations of relaxin, progesterone, and different inhibitors of PDE as described above. For the determination of hPRL release, 1 ml conditioned ESC medium was stored at -80 C until use, and hPRL content was later determined in the undiluted medium using a conventional ELISA format. This employed an hPRL-specific rabbit polyclonal antibody, previously validated for RIA (24), with a biotinylated hPRL tracer and horseradish peroxidase-conjugated streptavidin as detection system. hPRL standards (prepared in the laboratory of Dr. H. G. Friesen, University of Manitoba, Manitoba, Canada) were dissolved in basal ESC culture medium (as described above) plus 0.001% DMSO. The binding capacity of the ELISA in conditioned medium was approximately 93% of the values determined in standard E-PBS [0.1 M sodium phosphate (pH 7.0), 0.15 M NaCl, 5 mM EDTA, 0.2% BSA, and 0.01% thimerosal]. The preparation of the biotinylated hPRL tracer as well as the complete assay design followed the procedure described for a relaxin-ELISA (25). In detail, immunomodules (Nunc module 469949, coated with goat antirabbit {gamma}-globulin) were removed from the freezer and equilibrated to room temperature. E-PBS buffer (375 µl/well) was incubated for 2 min at room temperature and decanted. In the first incubation step, 50 µl sample and then 100 µl hPRL antibody [Ref. 24 ; dilution, 1:70,000 in E-PBS buffer containing 0.0005% (wt/vol) methyl orange] were pipetted into the wells and kept at 4 C for 18–24 h in the dark. In the second incubation step, 50 µl tracer solution [biotinylated hPRL at 12 ng/ml in E-PBS containing 0.0005% (wt/vol) bromphenyl blue] were added to the wells and incubated for an additional 2 h at 4 C. After decanting, 200 µl horseradish peroxidase-streptavidin solution (150 ng/ml in E-PBS) were added to the wells and incubated for 30 min at 4 C in the dark. Assay plates were washed four times with 375 µl cold wash solution (0.02% Tween 20 and 0.5% NaCl)/well. Empty wells were then filled with 250 µl of a freshly prepared substrate solution [100 mM sodium acetate and 5 mM citric acid (pH 5.5) supplemented with 20 µl/ml 0.02% (vol/vol) H2O2 and 20 µl 0.5% (wt/vol) 3,3',5,5'-tetramethylbenzidine in DMSO] and incubated for 40 min at room temperature in the dark. The reaction was stopped with 50 µl H2SO4/well, and the absorbance was measured at 450 nm. Sensitivity was in the range 0.5–81 ng/ml (equivalent to 25–4050 pg/well). The slope factor of the calibration curve was -1.16, and the EC50 was 5.06 ng hPRL/ml. Intra- and interassay coefficients of variants were 6.2% and 9.7%, respectively.

cAMP PDE assay

Confluent cultures of ESC in 92X17 TC dishes (Nunc) were washed twice in cold PBS, and cells were harvested by scraping in ice-cold PBS. After centrifugation (200 x g, 4 min) cell pellets were resuspended in homogenization buffer [10 mM HEPES (pH 7.4), 1 mM EDTA, and 1 mM dithiothreitol] supplemented with a protease inhibitor cocktail (Complete, Roche, Mannheim, Germany). Cell suspensions were allowed to swell on ice for 8 min and then Dounce homogenized (Kontes Co., Vineland, NJ) with 30 strokes. After addition of sucrose to a final concentration of 0.25 M, the cell lysate was centrifuged at 1600 x g for 10 min at 4 C. The supernatant was then frozen and stored immediately at -80 C until use. Protein concentration was determined using the Bio-Rad Laboratories (Munich, Germany) kit, with BSA (fraction V, Sigma-Aldrich Corp.) as standard. PDE activity was determined according to the method described by Stringfield and Morimoto (26) with modifications. Extracts of ESC (0.6 mg/ml protein, final concentration) were incubated at 30 C in a final concentration of 20 mM HEPES (pH 7.4), containing 90 mM KCl, 5 mM MgCl2, 0.75 mM CaCl2, and 1 µM cAMP in the presence or absence 10 µM of the PDE inhibitors IBMX, papaverine, rolipram, Ro-20-1724, dipyridamole, zaprinast, 8-methoxy-IBMX, erythro-9(2-hydroxy-3-nonyl)adenine (EHNA), cilostamide. All PDE inhibitors were purchased from Calbiochem, except papaverine (Sigma-Aldrich Corp.), and stocks were dissolved in DMSO before further dilution and application. The diluted DMSO (final concentration, 0.001%) was shown to have no effect on PDE activity. Reactions were terminated after 60 min by acidifying with HClO4 (final concentration, 0.4 M). After neutralization with a 1/6th volume of saturated KHCO3 solution, cAMP concentrations were measured by FIA on acetylated and diluted samples.

RT-PCR analysis

Total RNA was extracted from ESC of each experimental condition using RNeasy kits (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. cDNA was generated from 5 µg total RNA using 0.5 µg oligo(deoxythymidine) primer (Invitrogen, Karlsruhe, Germany) in a total volume of 12 µl. After denaturing at 70 C for 10 min with immediate cooling, the solution was supplemented with first strand buffer (Invitrogen), 20 mM (end concentration) of each deoxy-NTP (Genecraft, Munster, Germany), and 0.1 M dithiothreitol (Invitrogen) and preincubated for 2 min at 42 C. This was followed by 200 U Superscript II reverse transcriptase (Invitrogen) to give a total volume of 20 µl, and incubation was continued for a further 30 min. Reactions were stopped by heating at 70 C for 10 min and were stored at -20 C. Preparations without reverse transcriptase were used as negative controls, in which the absence of PCR products indicated a complete lack of contaminating genomic DNA.

PCRs were performed in a 50-µl total reaction volume, containing 1 µl cDNA, 200 nM each of sense and antisense primers, 0.1 U BioTherm-Red DNA polymerase (Genecraft), and 200 µM of each deoxy-NTP in 1x buffer (Biotherm, Genecraft). Primers for each PDE4 subtype (Table 1Go) were those used by Leroy et al. (27) (PDE4A and PDE4C) and Erdogan and Houslay (28) (PDE4B and PDE4D). Primers for hPRL and IGF-BP1 transcripts were described by Brosens et al. (29), and those for PDE8, PDE8A, PDE8B, LGR7, and glyceraldehyde-3-phosphate dehydrogenase (GAPD) are described in Table 1Go. Before amplification, samples were denatured at 95 C for 5 min. The amplification profile for each specific PCR consisted of denaturation at 94 C, annealing at the specific temperature, and extension at 72 C, as given in Table 2Go, followed by a final extension for 10 min at 72 C. The identity of all PCR products was confirmed by DNA sequencing using the dideoxy termination procedure. A 20-µl aliquot of each reaction mixture was electrophoresed on a 1% agarose gel and visualized by ethidium bromide fluorescence using the ImaGo system (B&L Systems, Maarssen, The Netherlands). Band intensities were analyzed densitometrically using the ImageQuant 5.0 software package (Molecular Dynamics, Sunnyvale, CA), and results, initially measured as integrated peak volume, were expressed as a percentage of the untreated control (mean ± SE; n = 4).


View this table:
[in this window]
[in a new window]
  		TABLE 1. List of oligonucleotide sequences used in the PCRs

 

View this table:
[in this window]
[in a new window]
  		TABLE 2. Amplification conditions for the PCRs

 
Statistical analysis

All experiments were performed at least in triplicate, and where appropriate, data were compared by ANOVA, followed by a post hoc Newman-Keuls test. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of various PDE inhibitors on PDE activity in extracts of human ESC

In an initial set of experiments, endogenous PDE activity was measured in ESC extracts using a standard cAMP conversion assay, testing various PDE inhibitors for their abilities to influence cAMP metabolism. Figure 1Go illustrates experiments carried out with 10 µM of the specific inhibitors. This concentration was chosen to represent an amount close to that needed to completely inhibit the specific PDE without, however, cross-reacting with other PDEs (specific EC50 values generally <5 µM; cross-reactivity, >100 µM) (30). Higher concentrations (100 µM) were also used, but although they generally increased inhibition, there also appeared to be less PDE specificity. Although the specific PDE inhibitors 8-methoxy-IBMX, EHNA, cilostamide, rolipram, Ro-20-1724, dipyridamole, and zaprinast, have comparable EC50 values for their specific substrates of less than 5 µM, the EC50 values of the nonspecific inhibitors IBMX and papaverine are approximately 10-fold higher. Therefore, the effects of IBMX and papaverine were also analyzed at 100 µM. At this concentration, both general inhibitors produce effects that are only marginally greater than those of the specific PDE4 inhibitors rolipram and Ro-20-1724 at the low concentration of 10 µM, indicating that PDE4 probably constitutes the major cAMP-degrading activity in ESC homogenates. Dipyridamole, which inhibits both PDE5 and PDE8, also showed significant inhibition. As the specific PDE5 inhibitor zaprinast was without marked effect, dipyridamole is probably acting at a PDE8 type of phosphodiesterase. In repeated experiments, the specific inhibitors of PDE1, PDE2, PDE3, and PDE5 showed no or only small (PDE3) effects. Although we cannot exclude that PDE1, -2, and -5 participate in cAMP degradation, these results suggest that both PDE4 and PDE8 are the more important PDE isoforms involved in cAMP catabolism in human endometrial stromal cells.



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 1. Pharmacological identification of PDE activities in extracts of human ESC. Endometrial cell homogenates were assayed for cAMP degradation using 1 µM cAMP as substrate. The effects of various PDE inhibitors were analyzed at 10 and 100 µM, as indicated. Untreated cell activity was used as the control. IBMX and papaverine were employed as nonspecific inhibitors, and zaprinast, 8-methoxy-IBMX (M-IBMX), EHNA, cilostamide, rolipram, Ro-20-1724, and dipyridamole were used as specific inhibitors of PDE5, -1, -2, -3, -4, and -5/8, respectively. Experiments with three independent cell batches from three different individuals gave essentially similar results. Asterisks indicate statistically significant differences (P < 0.05) from the control.

 
Effect of selected PDE inhibitors on relaxin-stimulated cAMP production by human endometrial stromal cells

The peptide hormone relaxin is specifically able to induce cAMP production in cultured endometrial stromal cells, presumably acting through the newly discovered 7TM receptor, LGR7 (31). This effect of relaxin can be moderately increased by addition (now to intact cells) of either IBMX or papaverine at high concentrations (e.g. 100 µM and 1 mM; Fig. 2Go). However, the basal level of cAMP is, as might be expected, also increased (Fig. 2Go, lower panel), such that the relative effect of relaxin is not changed (~1.5- to 2-fold). Addition of the PDE4-specific inhibitors rolipram and Ro-20-1724, at much lower concentrations (e.g. 10–100 µM; Fig. 2Go) resulted in a dramatic increase in cAMP production upon relaxin stimulation, which was supraproportional (~5-fold; for some batches of cells (not shown) this may be as much as 20- to 25-fold). Rolipram even appears to be effective at a concentration of only 1 µM (Fig. 2Go). This result strongly suggests that one of the isoforms of PDE4, more than other phosphodiesterases, is specifically involved in relaxin-induced production of cAMP in human ESC.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 2. Dose-response curves of various PDE inhibitors with (upper panel) or without (lower panel) relaxin at an optimal concentration (100 ng/ml) on the production of intracellular cAMP in ESC. Cultures of primary human ESC were preincubated for 30 min with various PDE inhibitors as indicated at concentrations ranging from 1 µM to 1 mM. Subsequently, relaxin was applied to all wells at 100 ng/ml for 20 min. The intracellular cAMP content was determined in triplicate wells within any one experiment, as illustrated. Experiments with three independent cell batches from three different individuals gave essentially similar results.

 
RT-PCR identification of PDE4, PDE8, and LGR7 mRNA in human ESC

Both PDE4 and PDE8 activity can be the result of the expression of different genetic isoforms. PDE4 activity is encoded by four related genes (PDE4A, -4B, -4C, and -4D) that can encode numerous isoforms through alternate splicing of RNA (32). PDE8 activity is encoded by two closely homologous genes (PDE8A and PDE8B) (33, 34). Transcript-specific PCR assays were developed using specific oligonucleotides capable of discriminating among PDE4A, -4B, -4C, and -4D mRNAs as well as between PDE8A and PDE8B mRNAs. In Fig. 3AGo, it can be seen that all PDE4 mRNAs are indeed expressed in human ESC. Interestingly, treatment of the cells for 3 d with 8Br-cAMP has no influence on PDE4A or PDE4B transcripts, but significantly up-regulates gene expression for PDE4C and PDE4D. In fact, PDE4D transcript levels are already significantly up-regulated after only 4 h of 8Br-cAMP treatment (not shown). PDE8B mRNA is the major PDE8 transcript expressed in ESC (Fig. 3BGo). PDE8A transcripts can be detected eventually after a second full round of PCR (not shown). In contrast, in the THP-1 cell line, which also responds to relaxin to produce an up-regulation of cAMP and is used here as control, PDE8B mRNA appears to be absent; instead, PDE8A transcripts are highly expressed. Confirming the recent cloning of the relaxin receptor (31), LGR7 transcripts were also identified in ESC (Fig. 3DGo). Treatment of ESC with 8Br-cAMP had no effect on the transcript levels of PDE8B and LGR7 (not shown). The identities of all PCR products were verified by DNA sequencing.



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 3. RT-PCRs specific for PDE4A-4D transcripts (A), PDE8 transcripts (C), and LGR7 transcripts (D). Primary human ESC were either decidualized by treatment with 1 mM 8Br-cAMP for 3 d (A) or left untreated before extraction of RNA and generation of cDNA. C and D, Aliquots of RT-PCR mixtures from the linear range of amplification were analyzed on an agarose gel and stained with ethidium bromide. B, Specific PCRs for the various PDE transcripts using cDNAs from four different individuals gave essentially similar results and were densitometrically quantified (four independent experiments from cell batches derived from four individual patients). Results (B) are expressed as a percentage of the respective untreated controls (mean ± SE; n = 4). As comparative internal standard in all experiments, GAPD mRNA was measured in parallel for all RNA extracts of treated and untreated cells and showed no effect of cAMP stimulation. Only PDE4C and PDE4D transcripts were significantly affected (P < 0.001) by cAMP stimulation. As indicated in C, PDE8 transcripts were assessed using either nondiscriminating primers or primer combinations specific for PDE8A or PDE8B. Neither PDE8 (C) nor LGR7 (D) transcripts showed any effect of cAMP stimulation (data not shown). C, RT-PCR products from ESC are compared with those from the THP-1 human monocyte cell line. A and D: c, Control reactions where cDNA has been omitted.

 
Effect of the PKA inhibitor, H89, on PDE4 activity

The effects of 8Br-cAMP to up-regulate the expression of PDE4C and PDE4D mRNAs after several hours are in keeping with the generally accepted short-term role of cAMP to activate PDE, thereby reducing high intracellular cAMP concentrations within minutes of agonist binding to a receptor. An elevated cAMP concentration activates PKA, which itself can activate several PDE4 subtypes by site-specific phosphorylation (35, 36, 37, 38). As a further verification of such a pathway, the specific PKA-inhibitor H89 was applied to ESC in the presence and absence of the effector relaxin, and the PDE4 inhibitor rolipram (Fig. 4Go). As expected, both relaxin and rolipram, individually and in synergy, caused a significant induction of cAMP. H89 caused a small, but not statistically significant, increase in cAMP generated in the presence of relaxin in the absence of rolipram, but with this PDE inhibitor, H89 increased cAMP production significantly (P < 0.01) in both the absence and presence of relaxin, thus supporting a PKA-dependent stimulatory effect on a PDE.



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 4. Effect of the PKA inhibitor H89 on relaxin-dependent (+) stimulation of cAMP production in ESC in the presence and absence of the specific PDE4 inhibitor rolipram. Intracellular cAMP concentrations were analyzed by specific FIA as described in Materials and Methods. Although treatment with 100 µM rolipram for 30 min indicated a marked synergy with relaxin in the controls, pretreatment for 60 min with the PKA inhibitor H89 had no significant effect with relaxin only, but showed a significant increase (P < 0.05) in cAMP production, both with and without relaxin, in the presence of rolipram. Data represent triplicate wells for a typical experiment. Experiments with three independent cell batches from three different individuals gave similar results.

 
Application of PDE4 inhibitors to induce decidualizaton in human ESC

Given the evidence of a role for PDE4 in human ESC, it is logical to determine whether longer-term (3-d) application of a PDE4 inhibitor (rolipram) has the same physiological effect as specifically increasing intracellular cAMP. Transcript levels for the PRL and IGF-BP1 genes, as markers of decidualization, were measured using semiquantitative RT-PCR. The identities of all PCR products were verified by DNA sequencing. Four completely independent batches of primary cells were assessed, and significance was estimated by ANOVA (Fig. 5Go). PRL mRNA can be induced by rolipram alone to a greater extent than by the hormone effector relaxin alone. Together, there is marked synergy of these two factors. Essentially similar results were observed for the mRNA for IGF-BP1, although the levels of transcript detected when relaxin or rolipram were added alone were very low. The synergy between both factors was nevertheless highly significant. Of interest here is that progesterone has no detectable effect, either alone or in conjunction with relaxin. Only when applied together with rolipram is a significant effect evident (P < 0.01), and then only for PRL mRNA, but not for IGF-BP1 mRNA. Papaverine, a more general PDE inhibitor, was also effective in synergizing with relaxin, although not to the same extent as rolipram. However, the EC50 for papaverine is approximately 10-fold greater than that for rolipram, but the use of very high concentrations (e.g. 1 mM) was precluded because of possible cytotoxicity in these long-term cultures. Similar experiments were carried out for up to 6 d (not shown) with very similar results.



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 5. RT-PCR analysis of the decidualization marker gene transcripts for PRL and IGF-BP1 in ESC cultured for 3 d in the absence or presence of progesterone, relaxin, and/or the PDE inhibitors papaverine (100 µM) and rolipram (100 µM). Total RNA was prepared from ESC that were untreated (control, vehicle only), treated with 1 mM 8Br-cAMP for 3 d (positive control), or treated with combinations of hormones and PDE inhibitors as indicated. Preliminary experiments were carried out to verify that all PCRs were within the linear range of amplification. Aliquots of RT-PCRs were analyzed on ethidium bromide-stained agarose gels (A). B, Results from four different experiments (cells from four patients) were densitometrically quantified using the ImageQuant 5.0 software package and are expressed as a percentage of the maximum intensity attained with any treatment within an experiment (mean ± SE; n = 4). Although for PRL mRNA this corresponded in all experiments with the 8Br-cAMP treatment, for IGF-BP1, maximum intensities were obtained uniformly with the relaxin plus rolipram treatment. Each bar represents the mean ± SEM for four different experiments, corresponding to cells derived from four different patients.

 
To confirm the decidualization phenotype using a different parameter, PRL peptide secretion into the culture medium was measured (Fig. 6Go). The results were very similar to those reporting specific gene transcript levels, with the PDE4 inhibitor rolipram being effective alone (P < 0.001), but showing marked synergy in conjunction with relaxin. Progesterone was generally lacking any effect, except in combination with rolipram and relaxin together (P < 0.001). In this system, papaverine had no statistically significant effect either alone or with relaxin. It should be noted that the combination of relaxin and rolipram is almost as effective as pure 8Br-cAMP alone. In these assays, relaxin alone or with progesterone was unable to increase the amount of PRL detectable in the culture medium. Measurement of intracellular cAMP contents paralleled the observed synergy (not shown).



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 6. A, PRL secretion into the medium of ESC cultured for 6 d in the absence or presence of progesterone (P; 0.1 µM), relaxin (RLX; 100 ng/ml), and/or the PDE inhibitors rolipram and papaverine (PAP), both at 100 µM. 8Br-cAMP (1 mM) was applied as a positive control to induce maximal PRL secretion. The PRL concentration in the medium was determined by a specific ELISA as described in Materials and Methods. The figure shows one typical experiment of four performed, which was repeated using different cell preparations from four different patients. All results were analyzed by ANOVA, followed by a post hoc Newman-Keuls test. Essentially similar results were evident already after 3 d of culture (data not shown). B, Concentration-dependent effect of rolipram on relaxin-mediated secretion of PRL from human ESC cultures. ESC were cultured for 4 d in the presence of rolipram with or without relaxin (100 ng/ml) as indicated at concentrations of the PDE inhibitor from 1–100 µM. All data were measured in triplicate within experiments; the figure shows results from three different experiments, using cells derived from three different patients, comparing the results as percentages of the maximum PRL production within an experiment. Maximum PRL production was consistently determined at 50 µM rolipram in the presence of relaxin.

 
In dose-response curves using rolipram (Fig. 6BGo), there was already a very marked effect on PRL production of the PDE inhibitor at the submaximal concentration of 10 µM. It should be noted that even at this concentration, rolipram alone, without relaxin, showed a significant (P < 0.01) effect on PRL secretion.

Finally, the cells in culture were checked for the morphological changes associated with decidualization as described in detail by Christian et al. (39). Cells were subjectively assessed on d 2, when the differences in differentiation rate between treatments was the most apparent (not shown). Although rolipram was generally applied at 100 µM, the typical morphological changes were already evident with only 10 µM rolipram when incubation times exceeded 2 d (not shown). In terms of the proportion of cells showing the typical cobblestone pattern of decidualized endometrial stromal cells compared with the relatively undifferentiated and fusiform mesenchymal cells at the start of culture, the various substances were effective in the order: vehicle < relaxin < rolipram < relaxin plus rolipram < 8Br-cAMP.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The peptide hormone relaxin has been shown to be a very effective agent with which to induce intracellular cAMP in ESC (40) and hence in vitro decidualization (16). The role of progesterone in this process is still unclear, although in vivo this steroid is known to be a key player (41, 42, 43). Relaxin is probably acting through the newly discovered 7TM receptor, LGR7 (31), which when overexpressed in transfected cells appears to couple to adenylate cyclase via Gs (31). Human ESC express transcripts encoding LGR7 (Fig. 3DGo), and Scatchard analyses estimate approximately 1000 relaxin-binding sites/ESC (44). Although the adenylate cyclase system might be involved in relaxin signaling (45), it has been suggested recently that the endogenous relaxin receptor couples only poorly to adenylate cyclase via Gs (46), and that an alternative, tyrosine kinase-dependent pathway may link receptor activation to a still uncharacterized PDE (20). This is based on the ability of a select group of tyrphostins, usually considered specific for the epidermal growth factor receptor tyrosine kinase or the platelet-derived growth factor receptor tyrosine kinase, to completely inhibit relaxin-induced cAMP production in human ESC and THP1 cells (20). Furthermore, unlike conventional Gs-coupled receptors such as vasoactive intestinal polypeptide, relaxin cannot induce adenylate cyclase activation in a dose-dependent manner in membrane preparations of ESC (20). Finally, relaxin-induced cAMP production can be inhibited by the MAPK inhibitors PD98059 and U0126 (21). All of these points suggest that activation of endogenous LGR7 is atypical, with only weak coupling to adenylate cyclase, and that another pathway, probably using a MAPK cascade, is involved. It was postulated that this MAPK pathway might, in fact, cause an inhibition of a PDE, as has been shown for the similar pathway activated by epidermal growth factor, which induces an inhibitory phosphorylation of PDE4D3 at Ser579 (47).

In this context, the present study set out to evaluate the complement of PDEs in human endometrial stromal cells and their possible role in relaxin-induced in vitro decidualization. RT-PCR as well as studies using specific PDE inhibitors suggest that all four PDE4 genes are expressed together with that for PDE8B. Elevation of intracellular cAMP leads to a further significant induction of PDE4C and PDE4D mRNA only, but not of the other identified members. This is similar to what has been reported for human myometrial cells, where 8Br-cAMP can also up-regulate PDE4D mRNA after about 5 h (48). The up-regulation of certain PDE4 variants in response to prolonged increases in cAMP has been discussed as a PDE-mediated desensitization to the effects of adenylate cyclase activators (35, 36). The fact that IBMX and papaverine have only modest effects on the cAMP-generating system, with rolipram or Ro-20-1724 being equally effective, suggests that the PDEs active in these cells are relatively insensitive to IBMX or papaverine, but selectively respond to the more specific inhibitors, such as rolipram, Ro-20-1724, or dipyrimadole. However, as IBMX is also an adenosine receptor antagonist, the blockade of endogenous adenosine effects could be at least partially responsible for its relatively poor response compared with the other inhibitors, which are weaker adenosine receptor antagonists (49). Dipyrimadole inhibits both PDE5 and PDE8, but as the specific PDE5 inhibitor zaprinast has only negligible effects, we can assume that dipyrimadole is targeting the PDE8B identified in these cells.

Treatment of cultured ESC with the PKA inhibitor H89 had significant, although relatively weak, effects on PDE activity, in contrast to the very pronounced effects reported for other cell systems (50). This suggests that short-term, PKA-dependent feedback inhibition of cAMP levels by activation of PDEs probably plays only a marginal role in ESC, consistent with the sustained cAMP response that is characteristic of decidualization.

Given the observed complement of PDEs in ESC, as described above, it was important to determine whether targeted inhibition of PDE4 alone was capable of producing the full phenotype of in vitro decidualization. Inhibitors of PDE4 have shown promising effects in several disease models, including heart failure, depression, asthma, and myometrial quiescence (51, 52, 53, 54). In cultured ESC, the archetypal PDE4 inhibitor rolipram alone was indeed able to induce decidualization-specific genes, such as PRL and IGF-BP1, secretion of PRL peptide, and appropriate morphological appearance, already at relatively low (~10 µM) concentrations (Figs. 5Go and Go6, and data not shown). Addition of relaxin with rolipram caused a marked synergistic effect, which could not be further enhanced by the addition of progesterone. Although the simplest explanation would be to evoke relaxin as a Gs-dependent activator of adenylate cyclase (55), with PDEs such as PDE4 acting downstream of this, as outlined above, there are numerous independent pieces of information suggesting that alternative pathways might be implicated and that the activated relaxin receptor, LGR7, only weakly couples to adenylate cyclase. A similar conclusion was recently drawn for relaxin acting upon rat heart (46).

Thus, there is evidence for an alternative pathway, by which relaxin in a tyrosine kinase-dependent manner may itself be able to inhibit a relatively IBMX-insensitive PDE, thereby up-regulating intracellular cAMP. There is a physiological advantage to a pathway targeting a PDE, because it is the principal function of the stroma to become decidualized and to maintain that decidualized status over a long period, especially if pregnancy occurs. The alternative, whereby adenylate cyclase leads to activation of PKA and hence to a PKA-dependent up-regulation of PDE activity (35, 36, 37, 38, 56) would not allow the sustained accumulation of cAMP in these cells, which is essential for decidualization. In contrast, a simultaneous inhibition of PDE activity would.

A further factor to be considered here is that decidualization and progression of the secretory phase are also accompanied by local production of relaxin in the endometrium, both in vivo (57, 58) and in vitro (59), thus creating a paracrine feed-forward system driving intracellular endometrial cAMP production. This could possibly be additionally supplemented by hCG production from the young blastocyst, which has been shown to activate what might be a comparable, LH receptor-dependent pathway to that of relaxin in endometrial epithelial cells (60). Another aspect that needs to be considered here and that has not been addressed in the present study is a possible role for intracellular compartmentalization. It has been suggested that some PDEs may become localized within the cell via A-kinase anchor proteins (AKAPs), together with PKA, thereby promoting their PKA-dependent up-regulation (61, 62). It has also been shown that one consequence of relaxin stimulation of ESC is to cause a relocalization of protein kinase C from the cytoplasm to the plasma membrane (63). Moreover, the protein kinase C-binding protein RACK-1 can interact with PDE4D3 (64). Therefore, part of the atypical signaling pathway for relaxin in these cells might be to induce a compartmentalization of the involved signal transduction components, which would favor an inactivation of PDE and a persistent cAMP elevation.

Finally, the results presented here suggest that a selective PDE4 inhibitor, especially in conjunction with a relaxin receptor agonist, might prove to be a very useful supplement to support decidualization and hence implantation in subfertile women. In this context, relaxin production by ovarian granulosa cells has been reported to be predictive for in vitro fertilization-embryo transfer cycles (65), and a PDE5 inhibitor (sildenafil citrate) has already been promulgated to improve local uterine blood flow and hence endometrial development in in vitro fertilization patients (66), thereby to increase implantation and ongoing pregnancy rates (67). Similarly, a PDE4 inhibitor has been suggested to induce tocolytic effects (cAMP-dependent) in the myometrium (48, 53). Currently, an intravaginally applied PDE4 inhibitor would appear to be the most suitable to encourage decidualization and endometrial development. Particularly the new generation of more specific PDE4 inhibitors are indicated, because they demonstrate markedly reduced side-effects compared with rolipram (52, 68, 69, 70).


   Acknowledgments
 
Our thanks go to Dr. Birgit Gellersen for advice on the decidualization assay, to Dr. Ralph Telgmann for developing the LGR7-PCR and for helpful discussions together with Dr. Yvonne Pohnke throughout this study, to Dr. David Sherwood for the generous gift of porcine relaxin, and to Dr. Matthias Schumacher for his considerable support in the development of the different immunoassays. We especially thank Prof. Lindner, his staff. and patients at the ELIM-Krankenhaus (Hamburg, Germany) for their excellent cooperation in helping us to obtain tissue samples.


   Footnotes
 
This work was supported by the Deutsche Forschungsgemeinschaft (Grant Iv7/9-1) and in part by the Leidenberger-Müller-Stiftung (Hamburg, Germany).

Abbreviations: 8Br-cAMP, 8-Bromo-cAMP; DMSO, dimethylsulfoxide; EHNA, erythro-9(2-hydroxy-3-nonyl)adenine; E-PBS, Eagle’s PBS buffer; ESC, endometrial stromal cell; FIA, fluorometric immunoassay; GAPD, glyceraldehyde-3-phosphate dehydrogenase; hPRL, human PRL; IBMX, 3-isobutyl-1-methylxanthine; IGF-BP1, IGF-binding protein-1; PDE, phosphodiesterase; PKA, protein kinase A; PRL, prolactin.

Received March 20, 2003.

Accepted October 7, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Telgmann R, Gellersen B 1998 Marker genes of decidualization: activation of the decidual prolactin gene. Hum Reprod Update 4:472–479[Abstract/Free Full Text]
  2. Irwin JC, de las Fuentes L, Dsupin BA, Giudice LC 1993 Insulin-like growth factor regulation of human endometrial stromal cell function: coordinate effects on insulin-like growth factor binding protein-1, cell proliferation and prolactin secretion. Regul Pept 48:165–177[CrossRef][Medline]
  3. Seppala M, Koistinen R, Rutanen E-M 1994 Uterine endocrinology and paracrinology: insulin-like growth factor binding protein-1 and placental protein 14 revisited. Hum Reprod 9:917–925[Abstract/Free Full Text]
  4. Stewart DR, Erikson MS, Erikson ME, Nakajima ST, Overstreet JW, Lasley BL, Amento EP, Seppala M 1997 The role of relaxin in glycodelin secretion. J Clin Endocrinol Metab 82:839–846[Abstract/Free Full Text]
  5. Wilcox AJ, Baird DD, Weinberg CR 1999 Time of implantation of the conceptus and loss of pregnancy. N Engl J Med 340:1796–1799[Abstract/Free Full Text]
  6. Paria BC, Reese J, Das SK, Dey SK 2002 Deciphering the cross-talk of implantation: advances and challenges. Science 296:2185–2188[Abstract/Free Full Text]
  7. Lindhard A, Bentin-Ley U, Ravn V, Islin H, Hviid T, Rex S, Bangsboll S, Sorensen S 2002 Biochemical evaluation of endometrial function at the time of implantation. Fertil Steril 78:221–233[CrossRef][Medline]
  8. Gonen Y, Casper RF 1991 Prediction of implantation by the sonographic appearance of the endometrium during controlled ovarian stimulation for in vitro fertilization (IVF). J In Vitro Fertil Embryo Transfer 7:146–152
  9. Sher G, Herbert C, Maassarani G, Jacobs MH 1991 Assessment of the late proliferative phase endometrium by ultrasonography in patients undergoing in-vitro fertilization and embryo transfer (IVF/ET). Hum Reprod 6:232–237
  10. Noyes N, Liu HC, Sultan K, Schattman G, Rosenwaks Z 1995 Endometrial thickness appears to be a significant factor in embryo implantation in in-vitro fertilization. Hum Reprod 10:919–922[Abstract/Free Full Text]
  11. Soliman S, Daya S, Collins J, Hughes EG 1994 The role of luteal phase support in infertility treatment: a meta-analysis of randomized trials. Fertil Steril 61:1068–1076[Medline]
  12. Pritts EA, Atwood AK 2002 Luteal phase support in infertility treatment: a meta-analysis of the randomized trials. Hum Reprod 17:2287–2299[Abstract/Free Full Text]
  13. Daley DC, Maslar IA, Rosenberg SM, Tohan N, Riddick DH 1981 Prolactin production by luteal phase defect endometrium. Am J Obstet Gynecol 140:587–591[Medline]
  14. Bulun SE, Noble LS, Takayama K, Michael MD, Agarwal V, Fisher C, Zhao Y, Hinshelwood MM, Ito Y, Simpson ER 1997 Endocrine disorders associated with inappropriately high aromatase expression. J Steroid Biochem Mol Biol 61:133–139[CrossRef][Medline]
  15. Brosens J, Brosens I 1998 Adenomyosis, In: Cameron I, FraserI, Smith S, eds. Clinical disorders of the menstrual cycle and endometrium. New York: Oxford University Press; 297–312
  16. Huang JR, Tseng L, Bischof P, Janne OA 1987 Regulation of prolactin production by progestin, estrogen, and relaxin in human endometrial stromal cells. Endocrinology 121:2011–2017[Abstract/Free Full Text]
  17. Tabanelli S, Tang B, Gurpide E 1992 In vitro decidualization of human endometrial stromal cells. J Steroid Biochem Mol Biol 42:337–344[CrossRef][Medline]
  18. Tang B, Gurpide E 1993 Direct effect of gonadotropins on decidualization of human endometrial stroma cells. J Steroid Biochem Mol Biol 47:115–121[CrossRef][Medline]
  19. Tang B, Guller S, Gurpide E 1993 Cyclic adenosine 3', 5'-monophosphate induces prolactin expression in stromal cells isolated from human proliferative endometrium. Endocrinology 133:2197–2203[Abstract/Free Full Text]
  20. Bartsch O, Bartlick B, Ivell R 2001 Relaxin signalling links tyrosine phosphorylation to phosphodiesterase and adenylyl cyclase activity. Mol Hum Reprod 7:799–809[Abstract/Free Full Text]
  21. Bartsch O, Bartlick B, Ivell R 2001 Relaxin signal transduction couples tyrosine phosphorylation to cAMP upregulation. In: Tregear GW, Ivell R, Bathgate RA, Wade JD, eds. Relaxin 2000. Dordrecht: Kluwer; 309–315
  22. Gellersen B, Kempf R, Telgmann R, DiMattia GE 1994 Nonpituitary human prolactin gene transcription is independent of Pit-1 and differentially controlled in lymphocytes and in endometrial stroma. Mol Endocrinol 8:356–373[Abstract/Free Full Text]
  23. Zarreh-Hoshyari-Khah R, Bartsch O, Einspanier A, Pohnke Y, Ivell R 2001 Bioactivity of recombinant prorelaxin from the marmoset monkey. Regul Pept 97:139–146[CrossRef][Medline]
  24. Gellersen B, DiMattia GE, Friesen HG, Bohnet HG 1989 Regulation of prolactin secretion in the human B-lymphoblastoid cell line IM-9-P3 by dexamethasone but not other regulators of pituitary prolactin secretion. Endocrinology 125:2853–2861[Abstract/Free Full Text]
  25. Einspanier A, Nubbemeyer R, Schlote S, Schumacher M, Ivell R, Fuhrmann K, Marten A 1999 Relaxin in the marmoset monkey: secretion pattern in the ovarian cycle and early pregnancy. Biol Reprod 1999 61:512–520[Abstract/Free Full Text]
  26. Stringfield TM, Morimoto BH 1997 Modulation of cyclic AMP levels in a clonal neural cell line by inhibitors of tyrosine phosphorylation. Biochem Pharmacol 53:1271–1278[CrossRef][Medline]
  27. Leroy MJ, Mehats C, Duc-Goiran P, Tanguy G, Robert B, Dallot E, Mignot TM, Grange G, Ferre F 1999 Effect of pregnancy on PDE4 cAMP-specific phosphodiesterase messenger ribonucleic acid expression in human myometrium. Cell Signalling 11:31–37[CrossRef][Medline]
  28. Erdogan S, Houslay MD 1997 Challenge of human Jurkat T-cells with the adenylate cyclase activator forskolin elicits major changes in cAMP phosphodiesterase (PDE) expression by up-regulating PDE3 and inducing PDE4D1 and PDE4D2 splice variants as well as down-regulating a novel PDE4A splice variant. Biochem J 321:165–175
  29. Brosens JJ, Takeda S, Acevedo CH, Lewis MP, Kirby PL, Symes EK, Krausz T, Purohit A, Gellersen B, White JO 1996 Human endometrial fibroblasts immortalized by simian virus 40 large T antigen differentiate in response to a decidualization stimulus. Endocrinology 137:2225–2231[Abstract]
  30. Soderling SH, Bayuga SJ, Beavo JA 1998 Cloning and characterization of a cAMP-specific cyclic nucleotide phosphodiesterase. Proc Natl Acad Sci USA 95:8991–8996[Abstract/Free Full Text]
  31. Hsu SY, Nakabayashi K, Nishi S, Kumagai J, Kudo M, Sherwood OD, Hsueh AJ 2002 Activation of orphan receptors by the hormone relaxin. Science 295:671–674[Abstract/Free Full Text]
  32. Houslay MD, Sullivan M, Bolger GB 1998 The multienzyme PDE4 cyclic adenosine monophosphate-specific phosphodiesterase family: intracellular targeting, regulation, and selective inhibition by compounds exerting anti-inflammatory and antidepressant actions. Adv Pharmacol 44:225–342
  33. Fisher DA, Smith JF, Pillar JS, St Denis SH, Cheng JB 1998 Isolation and characterization of PDE8A, a novel human cAMP-specific phosphodiesterase. Biochem Biophys Res Commun 246:570–577[CrossRef][Medline]
  34. Hayashi M, Matsushima K, Ohashi H, Tsunoda H, Murase S, Kawarada Y, Tanaka T 1998 Molecular cloning and characterization of human PDE8B, a novel thyroid-specific isozyme of 3',5'-cyclic nucleotide phosphodiesterase. Biochem Biophys Res Commun 250:751–756[CrossRef][Medline]
  35. Sette C, Iona S, Conti M 1994 The short-term activation of a rolipram-sensitive, cAMP-specific phosphodiesterase by thyroid-stimulating hormone in thyroid FRTL-5 cells is mediated by a cAMP-dependent phosphorylation. J Biol Chem 269:9245–9252[Abstract/Free Full Text]
  36. Sette C, Conti M 1996 Phosphorylation and activation of a cAMP-specific phosphodiesterase by the cAMP-dependent protein kinase. Involvement of serine 54 in the enzyme activation. J Biol Chem 271:16526–16534[Abstract/Free Full Text]
  37. Ekholm D, Belfrage P, Manganiello V, Degerman E 1997 Protein kinase A-dependent activation of PDE4 (cAMP-specific cyclic nucleotide phosphodiesterase) in cultured bovine vascular smooth muscle cells. Biochim Biophys Acta 1356:64–70[Medline]
  38. Baillie G, MacKenzie SJ, Houslay, MD 2001 Phorbol 12-myristate 13-acetate triggers the protein kinase A-mediated phosphorylation and activation of the PDE4D5 cAMP phosphodiesterase in human aortic smooth muscle cells through a route involving extracellular signal regulated kinase (ERK). Mol Pharmacol 60:1100–1111[Abstract/Free Full Text]
  39. Christian M, Mak I, White JO, Brosens JJ 2002 Mechanisms of decidualization. Reprod Biomed Online 3:24–30
  40. Fei DT, Gross MC, Lofgren JL, Mora-Worms M, Chen AB 1990 Cyclic AMP response to recombinant human relaxin by cultured human endometrial cells: a specific and high throughput in vitro bioassay. Biochem Biophys Res Commun 170:214–222[CrossRef][Medline]
  41. Psychoyos A 1986 Uterine receptivity for nidation. Ann NY Acad Sci 476:36–39[Medline]
  42. Bergh PA, Navot D 1992 The impact of embryonic development and endometrial maturity on the timing of implantation. Fertil Steril 58:537–542[Medline]
  43. Edwards RG 1995 Physiological and molecular aspects of human implantation. Hum Reprod 10:1–13[Free Full Text]
  44. Osheroff PL, King KL 1995 Binding and cross-linking of 32P-labeled human relaxin to human uterine cells and primary rat atrial cardiomyocytes. Endocrinology 136:4377–4381[Abstract]
  45. Kuznetsova L, Plesneva S, Derjabina N, Omeljaniuk E, Petseva M 1999 On the mechanism of relaxin action: the involvement of adenylyl cyclase signallig system. Regul Pept 80:33–39[CrossRef][Medline]
  46. Kompa AR, Samuel CS, Summers RJ 2002 Inotropic responses to human gene 2 (B29) relaxin in a rat model of myocardial infarction (MI): effect of pertussis toxin. Br J Pharmacol 137:710–718[CrossRef][Medline]
  47. Hoffmann R, Baillie GS, MacKenzie SJ, Yarwood SJ, Houslay MD 1999 The MAP kinase ERK2 inhibits the cyclic AMP-specific phosphodiesterase HSPDE4D3 by phosphorylating it at Ser579. EMBO J 18:893–903[CrossRef][Medline]
  48. Mehats C, Tanguy G, Dallot E, Robert B, Rebourcet R, Ferre F, Leroy MJ 1999 Selective up-regulation of phosphodiesterase-4 cyclic adenosine 3', 5'-monophosphate (cAMP)-specific phosphodiesterase variants by elevated cAMP content in human myometrial cells in culture. Endocrinology 140:3228–3237[Abstract/Free Full Text]
  49. Morgan AJ, Murray KJ, Challis RA 1993 Comparision of the effect of isobutylmethylxanthine and phosphodiesterase-selective inhibitors on cAMP levels in SH-SY5Y neuroblastoma cells. Biochem Pharmacol 45:2373–2380[CrossRef][Medline]
  50. Oki N, Takahashi SI, Hidaka H, Conti M 2000 Short term feedback regulation of cAMP in FRTL-5 thyroid cells. Role of PDE4D3 phosphodiesterase activation. J Biol Chem 275:10831–10837[Abstract/Free Full Text]
  51. Stafford JA, Feldman PL 1996 Chronic pulmonary inflammation and other therapeutic applications ofr PDE IV inhibitors. In: Doherty SM, ed. Annual reports in medicinal chemistry. San Diego: Academic Press; 71–80
  52. Teixeira MM, Gristwood RW, Cooper N, Hellewell PG 1997 Phosphodiesterase (PDE) 4 inhibitors: anti-inflammatory drugs of the future? Trends Pharmacol Sci 18:164–170[Medline]
  53. Dyke HJ, Montana JG 2002 Update on the therapeutic potential of PDE4 inhibitors. Expert Opin Invest Drugs 11:1–13
  54. Mehats C, Tanguy G, Paris B, Robert B, Pernin N, Ferre F, Leroy MJ 2000 Pregnancy induces a modulation of the cAMP phosphodiesterase 4-conformers ratio in human myometrium: consequences for the utero-relaxant effect of PDE4-selective inhibitors. J Pharmacol Exp Ther 292:817–823[Abstract/Free Full Text]
  55. Nguyen BT, Yang L, Sanborn BM, Dessauer CW 2003 Phosphoinositide 3-kinase activity is required for biphasic stimulation of cyclic AMP by relaxin. Mol Endocrinol 17:1075–1084[Abstract/Free Full Text]
  56. Persani L, Lania A, Alberti L, Romoli R, Mantovani G, Filetti S, Spada A, Conti M 2000 Induction of specific phosphodiesterase isoforms by constitutive activation of the cAMP pathway in autonomous thyroid adenomas. J Clin Endocrinol Metab 85:2872–2878[Abstract/Free Full Text]
  57. Bryant-Greenwood GD, Rutanen EM, Partanen S, Coelho TK, Yamamoto SY 1993 Sequential appearance of relaxin, prolactin and IGFBP-1 during growth and differentiation of the human endometrium. Mol Cell Endocrinol 95:23–29[CrossRef][Medline]
  58. Bogic LV, Mandel M, Bryant-Greenwood GD 1995 Relaxin gene expression in human reproductive tissues by in situ hybridization. J Clin Endocrinol Metab 80:130–137[Abstract]
  59. Palejwala S, Tseng L, Wojtczuk A, Weiss G, Goldsmith LT 2002 Relaxin gene and protein expression and its regulation of procollagenase and vascular endothelial growth factor in human endometrial cells. Biol Reprod 66:1743–1748[Abstract/Free Full Text]
  60. Srisuparp S, Strakova Z, Brudney A, Mukherjee S, Reierstad S, Hunzicker-Dunn M, Fazleabas AT 2003 Signal transduction pathways activated by chorionic gonadotropin in the primate endometrial epithelial cells. Biol Reprod 68:457–464[Abstract/Free Full Text]
  61. Dodge KL, Khouangsathiene S, Kapiloff MS, Mouton R, Hill EV, Houslay MD, Langeberg LK, Scott JD 2001 mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module. EMBO J 2001 20:1921–1930[CrossRef][Medline]
  62. Tasken KA, Collas P, Kemmner WA, Witczak O, Conti M, Tasken K 2001 Phosphodiesterase 4D and protein kinase a type II constitute a signaling unit in the centrosomal area. J Biol Chem 276:1999–2002
  63. Kalbag SS, Roginsky MS, Jelveh Z, Sulimovici S 1991 Phorbol ester, prolactin, and relaxin cause translocation of protein kinase C from cytosol to membranes in human endometrial cells. Biochim Biophys Acta 1094:85–91[Medline]
  64. Yarwood SJ, Steele MR, Scotland G, Houslay MD, Bolger GB 2001 The RACK1 signaling scaffold protein selectively interacts with the cAMP-specific phosphodiesterase PDE4D5 isoform. J Biol Chem 274:14909–14917
  65. Stewart DR, VandeVoort CA 1999 Relaxin secretion by human granulosa cell culture is predictive of in-vitro fertilization-embryo transfer success. Hum Reprod 14:338–344[Abstract/Free Full Text]
  66. Sher G, Fisch JD 2000 Vaginal sildenafil (Viagra): a preliminary report of a novel method to improve uterine artery blood flow and endometrial development in patients undergoing IVF. Hum Reprod 15:806–809[Abstract/Free Full Text]
  67. Sher G, Fish JD 2002 Effect of vaginal sildenafil on the outcome of in vitro fertilization (IVF) after multiple IVF failures attributed to poor endometrial development. Fertil Steril 78:1073–1076[CrossRef][Medline]
  68. Barnette MS, Underwood DC 2000 New phosphodiesterase inhibitors as therapeutics for the treatment of chronic lung disease. Curr Opin Pulm Med 6:164–169[CrossRef][Medline]
  69. Giembycz MA 2001 Cilomilast: a second generation phosphodiesterase 4 inhibitor for asthma and chronic obstructive pulmonary disease. Expert Opin Investig Drugs 10:1361–1379[CrossRef][Medline]
  70. Keller TH, Bray-French K, Demnitz FW, Muller T, Pombo-Villar E, Walker C 2001 Synthesis and structure-activity relationship of N-arylrolipram derivatives as inhibitors of PDE4 isozymes. Chem Pharm Bull 49:1009–1017



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
R. Anand-Ivell, K. Heng, B. Hafen, B. Setchell, and R. Ivell
Dynamics of INSL3 Peptide Expression in the Rodent Testis
Biol Reprod, September 1, 2009; 81(3): 480 - 487.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
M. Sasseville, F. K. Albuz, N. Cote, C. Guillemette, R. B. Gilchrist, and F. J. Richard
Characterization of Novel Phosphodiesterases in the Bovine Ovarian Follicle
Biol Reprod, August 1, 2009; 81(2): 415 - 425.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
K. Heng, R. Ivell, P. Wagaarachchi, and R. Anand-Ivell
Relaxin signalling in primary cultures of human myometrial cells
Mol. Hum. Reprod., October 1, 2008; 14(10): 603 - 611.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
W. Yan, J. Chen, A. A Wiley, B. D Crean-Harris, F. F Bartol, and C. A Bagnell
Relaxin (RLX) and estrogen affect estrogen receptor {alpha}, vascular endothelial growth factor, and RLX receptor expression in the neonatal porcine uterus and cervix
Reproduction, May 1, 2008; 135(5): 705 - 712.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
A. Kern, D. Hubbard, A. Amano, and G. D. Bryant-Greenwood
Cloning, Expression, and Functional Characterization of Relaxin Receptor (Leucine-Rich Repeat-Containing G Protein-Coupled Receptor 7) Splice Variants from Human Fetal Membranes
Endocrinology, March 1, 2008; 149(3): 1277 - 1294.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
O.P.M. Smith, H.N. Jabbour, and H.O.D. Critchley
Cyclooxygenase enzyme expression and E series prostaglandin receptor signalling are enhanced in heavy menstruation
Hum. Reprod., May 1, 2007; 22(5): 1450 - 1456.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
T. Kajihara, M. Jones, L. Fusi, M. Takano, F. Feroze-Zaidi, G. Pirianov, H. Mehmet, O. Ishihara, J. M. Higham, E. W.-F. Lam, et al.
Differential Expression of FOXO1 and FOXO3a Confers Resistance to Oxidative Cell Death upon Endometrial Decidualization
Mol. Endocrinol., October 1, 2006; 20(10): 2444 - 2455.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
S. Hombach-Klonisch, J. Bialek, B. Trojanowicz, E. Weber, H.-J. Holzhausen, J. D. Silvertown, A. J. Summerlee, H. Dralle, C. Hoang-Vu, and T. Klonisch
Relaxin Enhances the Oncogenic Potential of Human Thyroid Carcinoma Cells
Am. J. Pathol., August 1, 2006; 169(2): 617 - 632.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
J. Francis, R. Rai, N. J. Sebire, S. El-Gaddal, M. S. Fernandes, P. Jindal, A. Lokugamage, L. Regan, and J. J. Brosens
Impaired expression of endometrial differentiation markers and complement regulatory proteins in patients with recurrent pregnancy loss associated with antiphospholipid syndrome
Mol. Hum. Reprod., July 1, 2006; 12(7): 435 - 442.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
R. A. Bathgate, R. Ivell, B. M. Sanborn, O. D. Sherwood, and R. J. Summers
International Union of Pharmacology LVII: Recommendations for the Nomenclature of Receptors for Relaxin Family Peptides.
Pharmacol. Rev., March 1, 2006; 58(1): 7 - 31.
[Abstract] [Full Text] [PDF]

Home page
 Clin Med ResHome page
C. S. Samuel
Relaxin: Antifibrotic Properties and Effects in Models of Disease
Clin. Med. Res., November 1, 2005; 3(4): 241 - 249.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
E. Dimitriadis, C. Stoikos, M. Baca, W. D. Fairlie, J. E. McCoubrie, and L. A. Salamonsen
Relaxin and Prostaglandin E2 Regulate Interleukin 11 during Human Endometrial Stromal Cell Decidualization
J. Clin. Endocrinol. Metab., June 1, 2005; 90(6): 3458 - 3465.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
M. Tang, J. Mazella, H. Hui Zhu, and L. Tseng
Ligand activated relaxin receptor increases the transcription of IGFBP-1 and prolactin in human decidual and endometrial stromal cells
Mol. Hum. Reprod., April 1, 2005; 11(4): 237 - 243.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bartsch, O.
Right arrow Articles by Ivell, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bartsch, O.
Right arrow Articles by Ivell, R.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals