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Endocrinology Vol. 144, No. 4 1284-1291
Copyright © 2003 by The Endocrine Society

ARTICLE

Prostaglandin (PG) FP and EP1 Receptors Mediate PGF2{alpha} and PGE2 Regulation of Interleukin-1ß Expression in Leydig Cell Progenitors

Laurence Walch, Emanuela Clavarino and Patricia L. Morris

Population Council (L.W., E.C., P.L.M.) and The Rockefeller University (P.L.M.), New York, New York 10021

Address all correspondence and requests for reprints to: Patricia L. Morris, Center for Biomedical Research, Population Council and The Rockefeller University, 1230 York Avenue, New York, New York 10021. E-mail: p-morris{at}popcbr.rockefeller.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prostaglandins (PG) mediate IL-1ß regulation of several interleukin mRNAs in progenitor Leydig cells. PGE2 and PGF2{alpha} potently reverse indomethacin (INDO; a cyclooxygenase inhibitor) inhibition of IL-1ß autoinduction. IL-1ß increases PGE2 and PGF2{alpha} production. To determine the PG receptors involved in this regulation, this study established by RT-PCR and Western analyses which specific receptors for PGE2 (EP receptors) and PGF2{alpha} (FP receptors) are expressed in progenitors. Pharmacological characterization of receptors involved in PGE2 and PGF2{alpha} regulation of IL-1ß mRNA levels was ascertained using real-time PCR analyses. FP, EP1, EP2, and EP4 receptor mRNAs and proteins, and an EP3 receptor subtype were detected. IL-1ß treatment (24-h) significantly decreased EP1 receptor levels; INDO abrogated this down-regulation. FP, EP2, and EP4 receptor levels increased after IL-1ß and IL-1ß + INDO. A selective FP agonist, cloprostenol (0.1 µM), and PGF2{alpha} (10 µM) had similar effects on IL-1ß mRNA levels in progenitors treated with IL-1ß + INDO. None of the EP2/EP4 agonists [butaprost, misoprostol, or 11-deoxy PGE1 (10 µM)] affected IL-1ß mRNA levels. In contrast, EP1/EP3 agonists (17-phenyl trinor PGE2 and sulprostone) increased IL-1ß mRNAs in a dose-dependent manner. EP1 receptor subtype-selective antagonist, SC-51322, blocked IL-1ß-induced and [IL-1ß + INDO + 17-phenyl trinor PGE2]-induced increases in IL-1ß mRNAs. Taken together, our data demonstrate that FP and EP1 receptors mediate PGF2{alpha} and PGE2 induction of progenitor IL-1ß expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROSTAGLANDINS (PG), THE PRODUCTS of arachidonic acid cyclooxygenation, regulate a broad range of physiological processes, such as inflammation and immune responses (1). In particular, PGE2 and PGF2{alpha} are crucial regulators of female reproduction (2). The biological activities of PG are mediated by the activation of specific cell surface receptors. PGE2 receptors (EP receptors) are pharmacologically distinguished into four different subtypes (EP1–EP4), each receptor encoded by a distinct gene and differently expressed in diverse target cells (3, 4). EP receptors differentially couple to various guanine-nucleotide binding proteins and downstream signal transduction systems (3, 4). EP1 receptors activate phospholipase C and phosphatidylinositol turnover and stimulate the release of intracellular calcium. EP2 and EP4 receptors activate adenylate cyclase. EP3 receptors are generally associated with diminished levels of cAMP and stimulation of calcium release, although a number of splice variants of these receptors coupled to different guanine-nucleotide binding proteins have been described. PGF2{alpha} receptors (FP receptors) as well as EP1 and EP3 receptors are coupled with the mobilization of intracellular calcium (3, 4).

IL-1ß induces the production of PG by up-regulation of the inducible isoform of cyclooxygenase (COX) 2 in a broad array of cell types and tissues (5). In the adult testis, Leydig cells are devoted to the production of testosterone, the hormone essential for spermatogenesis. Khan et al. (6) previously showed that IL-1ß stimulates DNA synthesis in Leydig cells isolated from prepubertal rats, e.g. differentiating immature Leydig cells, but not in adult Leydig cells. Furthermore, IL-1ß was shown to increase LH-induced steroidogenesis in immature rat Leydig cells (7). Population of the testicular interstitium by macrophages results in a close association of resident macrophages with Leydig cells during prepubertal development. Detection of IL-1-like activity in the testis is observed at 20 d of age, coinciding with the development and differentiation of immature Leydig cells. It has been proposed that IL-1 plays a physiological role in the paracrine/autocrine regulation of pubertal development of Leydig cells (8, 9, 10). Whether the effects of IL-1ß on pubertal Leydig cells are mediated by PG was not investigated in earlier studies. Overall, the precise role of PG in the male reproductive system is poorly understood, although the isoforms of COX are present in the developing male reproductive tract (11).

We previously showed that IL-1ß is a potent inducer of several interleukins (namely, IL-1{alpha}, IL-1ß, and IL-6) in progenitor Leydig cells (PLC) isolated from 18-d-old rats (12). Furthermore, our studies demonstrated that the COX2 pathway is involved in this regulation because the nonsteroidal antiinflammatory drugs indomethacin (INDO; a nonselective COX1 and COX2 inhibitor) and NS-398 (a selective COX2 inhibitor) significantly impaired induction of IL expression by IL-1ß. In PLC treated with IL-1ß and INDO, the exogenous PGF2{alpha} and PGE2 are the most potent PG that restore the level of IL-1ß mRNA to that achieved in absence of INDO. Measurements of PG by ELISA showed that IL-1ß treatment significantly increased PLC production of multiple PG (12). PGE2 and, to a lesser extent, PGF2{alpha} were the major PG released by PLC treated with IL-1ß. The aim of the present study was to determine which PG receptors and their subtypes are expressed in progenitors. In addition, we undertook the pharmacological characterization of the PG receptors involved in the PGE2 and PGF2{alpha} regulation of IL-1ß mRNA levels in PLC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell preparations
PLC were prepared from 18-d-old Sprague-Dawley (SD) rats [Crl: CD(SD)BR-CD, Charles River Laboratories, Inc., Kingston, NY] as previously described (12). Briefly, after Collagenase/Dispase (Roche Diagnostics, Mannheim, Germany) dissociation, a fraction enriched in PLC was isolated using a Percoll density gradient separation (Amersham Pharmacia Biotech, Uppsala, Sweden). The purity of the PLC preparations was assessed using 3{alpha}-hydroxysteroid dehydrogenase (3{alpha}-HSD) immunocytochemistry and 3ß-HSD histochemistry as previously described (13). On isolation and purification, the PLC from d 18 testes were macrophage free, 30 ± 1% lightly positive for 3ß-HSD, with the remaining cells strongly or weakly 3{alpha}-HSD positive (n = 8). Such progenitors were used immediately as freshly isolated or established in culture as previously reported (12).

The progenitors were cultured for 1 d in serum-free medium supplemented with 0.1% fetal bovine serum (JRH Biosciences, Lenexa, KS). PLC were rinsed twice with fresh serum-free and phenol red-free culture medium and then treated (24 h) with IL-1ß (10 ng/ml) in the absence or presence of INDO (10 µM). Cells were also concomitantly treated with prostaglandins (PGF2{alpha} or PGE2) or selective agonist for prostanoid receptors as indicated in Results. SC51322 was added to the culture medium 30 min before IL-1ß treatment. The doses of all drugs were selected on the basis of several similar studies that showed their maximal effects, specificity, and effectiveness (12, 14, 15). Adult Leydig cells were prepared from SD rats (age, 55–65 d) and purified by Percoll gradient and centrifugal elutriation as previously reported (13, 16). These purified adult rat Leydig cells were more than 98% 3ß-HSD-positive and macrophage-free. Procedures involving the use of animals strictly followed the Guidelines for the Care and Use of Laboratory Animals set forth by the National Institutes of Health. The immortalized MA-10 cell line (kindly provided by Dr. Mario Ascoli, University of Iowa, Iowa City, IA) used in this study was maintained as previously reported (13).

Cytokines, prostaglandins, and drugs
Recombinant mouse IL-1ß was purchased from R&D Systems (Minneapolis, MN). IL-1ß was reconstituted in a sterile stock solution of 0.1% BSA in PBS (final BSA concentration, 0.0001%). PGE2, PGF2{alpha}, 11-deoxy PGE1 (9-oxo-15S-hydroxy-prost-13E-en-1-oic acid), 17-phenyl trinor PGE2 (9-oxo-11{alpha},15S-dihydroxy-17-phenyl-18,19,20-trinor-prosta-5Z,13E-dien-1-oic acid), butaprost (9-oxo-11{alpha},16R-dihydroxy-17-cyclobutyl-prost-13E-en-1-oic acid, methyl ester), cloprostenol [9{alpha},11{alpha},15R-trihydroxy-16-(3-chlorophenoxy)-17,18,19,20-tetranor-prosta-5Z,13E-dien-1-oic acid, sodium salt], misoprostol (9-oxo-11{alpha},16-dihydroxy-16-methyl-prost-13E-en-1-oic acid, methyl ester), and sulprostone [N-(methylsulfonyl)-9-oxo-11{alpha},15R-dihydroxy-16-phenoxy-17,18,19,20-tetranor-prosta-5Z,13E-dien-1-amide] were purchased from Cayman Chemical (Ann Arbor, MI). SC-51322 [2-[3-[(2-furanylmethyl)-thiol]-1-oxopropyl]hydrazide] was from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). INDO [1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3-acetic acid)] was obtained from Sigma (St. Louis, MO). Prostaglandins, synthetic agonists, SC-51322, and INDO were dissolved in dimethylsulfoxide (DMSO), and subsequent dilutions were performed as needed in serum-free and phenol red-free sterile medium on the day of the experiment. DMSO alone was used as vehicle in all plates as required. Final DMSO concentrations varied between 0.3 and 0.8%. At these concentrations, DMSO did not affect IL-1ß mRNA levels compared with those in saline vehicle-treated controls.

Western analysis
Whole cell lysates were prepared from primary progenitors as follows: PLC were lysed in 200 µl of extraction buffer [10 mM Tris-HCl (pH 7.8), containing 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 2 µg/ml apoprotinin, 2 mg/ml leupeptin, 2 mg/ml pepstatin A, and 1 mM sodium orthovanadate (Sigma)] for 15 min on ice. Cellular debris were pelleted by centrifugation at 12,000 x g for 15 min at 4 C. Proteins were concentrated using Microcon Centrifugal Filter Devices (Millipore Corp., Bedford, MA) as recommended by the manufacturer. The protein concentration was determined by the Bradford assay. Samples (20 µg protein/well) were then subjected to SDS-PAGE using 4–20% Tris-glycine gel (Novex, San Diego, CA) and transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH). The membrane was sequentially probed with specific rabbit polyclonal antibodies for EP receptors (antimouse EP1 IgG, antirat EP2 IgG, antirat EP3 IgG, and antihuman EP4 IgG; Alpha Diagnostic International, San Antonio, TX), and FP receptors (antimouse FP IgG; Cayman Chemical). A monoclonal anti-ß-actin antibody was used to normalize protein loading to that of specific proteins in each lane (Sigma). Blots were developed using a goat antirabbit or mouse IgG antibody conjugated to horseradish peroxidase (Cell Signaling Technology, Beverly, MA) and the ECL Plus Western blotting detection reagents (Amersham Pharmacia Biotech, Buckinghamshire, UK).

Total RNA extraction
Total RNA was extracted from PLC using the Trizol reagent (Life Technologies, Inc., Grand Island, NY) according to the manufacturer’s instructions. RNA was measured using 260/280 UV spectrophotometry.

Real-time PCR
Real-time PCR (TaqMan) analysis was used for relative quantitation of mRNA levels using a standard curve method as previously described (12). The detection of IL-1ß mRNAs was performed using proprietary predeveloped TaqMan primers and FAM-labeled probe (PE Applied Biosystems, Foster City, CA). 18S Ribosomal RNA (VIC-labeled probe) was used to normalize the data.

TaqMan reactions were set up in optical 96-well reaction plates by adding 24 µl of a mix containing 12.5 µl of TaqMan Universal PCR Master Mix 2x, 2.5 µl of primers and probes (10x, according to the manufacturer’s instructions), and 9 µl of autoclaved water. One microliter of the reverse transcription (RT) samples prepared as described above was subsequently added to each well. The reactions were set up in triplicate.

Real-time fluorescence-monitored PCRs were performed using an Applied Biosystem Model 7700 Sequence Detection System. The temperature profile was as follows: 50 C for 2 min, 95 C for 10 min, then 95 C for 15 sec and 60 C for 1 min for 40 cycles. Using the manufacturer’s software, a threshold above the noise was chosen, and the cycle number at which fluorescence, generated by the cleavage of the probe, exceeded the threshold was determined for each well. For each real-time PCR analysis, a standard curve was generated by six 2-fold serial dilutions in water of the RT samples corresponding to the 24-h treatment of PLC with IL-1ß. The cycle number values were compared with the standard curve after linear regression analysis. Experimental samples were diluted 3-fold for comparison with the standard curve. A no-template control was performed for each reaction in duplicate.

RT-PCR
Total RNA (2 µg) was subjected to RT for 15 min at 42 C with 2.5 U/µl Moloney murine leukemia virus reverse transcriptase (PE Applied Biosystems). RT was performed in a 20-µl mixture containing 5 mM MgCl2, 1x PCR buffer II (PE Applied Biosystems), 4 mM of each deoxynucleotide triphosphate, 1 U/µl ribonuclease inhibitor, and 2.5 µM random hexamers. Samples were then denatured for 5 min at 99 C. A no-template control was performed for each experiment to establish the absence of genomic contamination of the samples.

PCR was performed by using 5 µl of each RT product as a template that was amplified in a 25-µl PCR using 25 mU AmpliTaq DNA Polymerase (PE Applied Biosystems). The following primers were used: EP1 sense primer, 5'-TGTATACTGCAGGACGTGCGCCC-3'; EP1 antisense primer, 5'-GGGCAGCTGTGGTTGAAGTGATG-3' (537-bp product; Ref. 17); EP2 sense primer, 5'-CCGCGCGTGTACCTATTTCGC-3'; EP2 antisense primer, 5'-GCTCCGAAGCTGCATGCGAA-3' (370-bp product; Ref. 18); EP3 sense primer, 5'-GCCGGGAGAGCAAACGCAAAAA-3'; EP3 antisense primer, 5'-ACACCAGGGCTTTGATGGTCGCCAGG-3' (534-bp product; Ref. 17); EP4 sense primer, 5'-TTCCGCTCGTGGTGCGAGTGTTC-3'; EP4 antisense primer, 5'-GAGGTGGTGTCTGCTTGGGTCAG-3' (424-bp product; Ref. 18); FP sense primer, 5'-GGCGTTTATCTCCACAAC-3'; FP antisense primer, 5'-CTAGATGCTTGCTGATT-3' (1104-bp product; Ref. 19). The amplification of the S16 ribosomal gene was used as an internal control with the following primers: 5'-CCCAGGTGTGACTTTGTCCT-3' and 5'-GACAAGACGAAGACCCGTT-3' (20).

The PCR mixture contained 2 mM MgCl2, 1x PCR buffer II, with each primer at 0.2 µM. Amplification was performed in a programmable thermal controller (MJ Research, Inc., Watertown, MA). The samples were first denatured at 95 C for 2 min, followed by 35 PCR cycles; the temperature profile was 95 C for 30 sec, annealing temperature for 30 sec, and 72 C for 90 sec. The annealing temperature for EP1, EP3, and EP4 was 61 C, and for EP2 and FP, it was 56 C. After the last cycle, an additional extension incubation of 7 min (72 C) was performed.

After amplification, PCR products (5 µl of each sample) were subjected to size separation by polyacrylamide gel (4–20% Tris/boric acid/EDTA gels; Novex, San Diego, CA). A set of DNA base pair-markers was electrophoresed alongside the samples to estimate product sizes (DNA Molecular Marker XIV, PE Applied Biosystems). After the gels were stained with ethidium bromide (2.5 µg/ml) for 15 min, the bands were visualized by UV fluorescence.

Sequencing
RT product (6 µl) was amplified in a total of a 75-µl PCR mixture using 2.5 U/µl AmpliTaq DNA Polymerase (PE Applied Biosystems). The PCR mixture contained 2 mM MgCl2, 1x PCR buffer II, with the particular primers at 0.2 µM. Amplification was performed in a programmable thermal controller (MJ Research, Inc.). The samples were first denatured at 95 C for 2 min, followed by 35 PCR cycles; the temperature profile was 95 C for 30 sec, 61 C for 30 sec, and 72 C for 90 sec. After the last cycle, an additional 7-min extension incubation (72 C) was performed.

Direct purification of each PCR product from separate amplification reactions and DNA cleanup from other enzymatic reactions were performed using QIAquick PCR Purification Kits (QIAGEN Inc., Valencia, CA). Purified PCR products were quantified using a {lambda} 35 spectrophotometer (Perkin-Elmer Corp., Shelton, CT). Sequencing was performed using 75 ng of each purified PCR product (The Rockefeller University DNA Sequencing Resource Center, New York, NY). Each resulting sequence was compared with the GenBank nucleotide database search using both National Center for Biotechnology Information standard nucleotide-nucleotide blast and Biology Workbench nucleotide blast (http://workbench.sdsc.edu/).

Data analysis
Densitometric analysis was performed using the PC version of NIH Image software (Scion Image; Scion Corp., Frederick, MD) after photography with a computer-assisted camera (Eastman Kodak, Rochester, NY). Densitometric analyses are expressed in arbitrary units. Replicate PLC controls for Western analyses are normalized to unit value. Real-time PCR data are expressed as a percentage of the IL-1ß 24-h response. All results are means ± SEM derived from the number of different experiments as indicated (n).

Statistical analyses were performed using Student’s t test, with a confidence level of 95%.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of EP receptors
EP1, EP2, EP3, EP4, and FP receptor mRNAs were detected using RT-PCR analyses. Total RNA was prepared from freshly isolated progenitors (Fig. 1Go, lane 1, PLC) and mature Leydig cells purified from adult rats (lane 2, ALC). A representative experiment with one of three sets of freshly isolated preparations is illustrated (Fig. 1Go). Sequencing confirmed the identity of the single PCR products. EP2 and FP mRNAs were not detected in adult rat Leydig cells.



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  		Figure 1. Expression of EP and FP receptors in rat progenitor and adult Leydig cells. PLC (lane 1) and adult Leydig cells (ALC, lane 2) were freshly isolated and purified as indicated in Materials and Methods. Total RNA was immediately prepared from separate experiments. The illustration shows RT-PCR analyses that were performed using the same RT from either PLC or ALC with each primer set for the EP and FP receptor mRNAs.

 
To next investigate which PG receptor subtypes are expressed in nonstimulated and IL-1ß-treated primary PLC in the presence or absence of INDO, immunoblot analyses were performed (Fig. 2Go). EP1, EP2, EP4, and FP receptor proteins were demonstrated in control PLC (Fig. 2Go, lane 1). In contrast to the other EP and FP receptors, EP3 receptors could not be detected by antirat EP3-immunoblot analysis in either freshly isolated or cultured rat PLC. However, the EP3 receptor protein was readily observed in extracts from the immortalized mouse Leydig cell line (MA-10) under the same conditions (data not shown). Sequencing of the EP3 PCR product amplified using RNA from freshly isolated, purified progenitors showed at least 93% sequence identities with those for the four known splicing variants for the EP3 receptor isoforms (21).



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  		Figure 2. EP and FP receptor proteins in rat Leydig progenitors. PLC were cultured for 24 h without (lane 1) or with IL-1ß (10 ng/ml) in the absence (lane 2) or presence of INDO (10 µM; lane 3). Whole cell lysates were prepared, and proteins were subjected to SDS-PAGE followed by repetitive immunoblot analysis on a given membrane for specific PG receptors and ß-actin. A, Representative sequential Western analysis using a single membrane. The sequence of blotting for separate experiments was varied, giving similar results for each receptor (data not shown). B, Densitometric analyses were performed for three replicates; specific bands for PG receptors were normalized to those for ß-actin and expressed in arbitrary units. *, A significant difference obtained in the presence of IL-1ß or IL-1ß + INDO compared with those of control (Ct) values (Student’s t test).

 
IL-1ß treatment significantly decreased EP1 receptor levels (Fig. 2AGo, lane 2; Fig. 2BGo, middle columns). In the presence of INDO (Fig. 2AGo, lane 3; Fig. 2BGo, right columns), this IL-1ß down-regulation was abrogated but remained decreased relative to control. FP, EP2, and EP4 receptor levels increased with IL-1ß and/or IL-1ß + INDO treatment (Fig. 2Go, A and B).

PG receptors involved in PGF2{alpha} and PGE2 induction of IL-1ß mRNA levels in progenitors
Cloprostenol, a selective FP receptor agonist, was used to evaluate the involvement of FP receptors in the up-regulation of IL-1ß mRNA levels. Data presented show that at a low dose, cloprostenol (0.1 µM) and PGF2{alpha} (10 µM) have a similar effect on IL-1ß mRNA levels in PLC treated with IL-1ß + INDO (Fig. 3Go).



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  		Figure 3. Effect of FP receptor agonists on steady state levels of IL-1ß mRNAs in progenitors treated with IL-1ß and INDO. PLC were cultured for 24 h without [control (Ct)] or with IL-1ß (10 ng/ml) in the absence or presence [vehicle (Veh)] of INDO (10 µM). PGF2{alpha} (10 µM) or cloprostenol (0.1 µM) were added concomitantly with IL-1ß + INDO. Total RNAs were extracted, and cDNAs were amplified by real-time RT-PCR using primers and probes for IL-1ß. Results are means ± SEM from (n) individual experiments. Data are normalized with 18S values and are expressed as percentage of IL-1ß 24-h response. *, A significant difference from similar values obtained in the presence of IL-1ß and INDO compared with control (Student’s t test).

 
Next, studies were performed to further evaluate the EP receptor subtype(s) involved in the PGE2 up-regulation of IL-1ß mRNA levels. Progenitors were treated for 24 h in the presence of IL-1ß + INDO using a panel of synthetic agonists that selectively activate one or more EP subtypes. As shown in Table 1Go, EP2 and EP4 agonists (10 µM) failed to enhance IL-1ß mRNA levels in PLC. In contrast, the EP1/EP3 agonists 17-phenyl trinor PGE2 and sulprostone increase the level of IL-1ß mRNAs in a dose-dependant manner (Fig. 4Go and Table 1Go). Misoprostol, an EP2/EP3 agonist (10 µM), was without effect (Table 1Go). Finally, SC-51322, an EP1 receptor subtype selective antagonist, dose-dependently blocked the IL-1ß and [IL-1ß + INDO + 17-phenyl trinor PGE2]-induced increase in IL-1ß mRNA levels (Fig. 5Go).


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  		Table 1. Effect of EP receptor agonists on steady state levels of IL mRNAs in PLC treated with IL-1ß and INDO

 


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  		Figure 4. Effect of EP1/EP3 receptor agonists on steady state levels of IL-1ß mRNAs in PLC treated with IL-1ß and INDO. PLC were cultured for 24 h with IL-1ß (10 ng/ml) and INDO (10 µM) in the absence [vehicle (Veh); n = 22] or presence of PGE2 (n = 4–14), sulprostone (n = 4–6), or 17-phenyl-trinor-PGE2 (at different doses; n = 4–11). Total RNAs were extracted, and cDNAs were amplified by real-time RT-PCR using primers and probes for IL-1ß. Results are means ± SEM from (n) individual experiments. Data are normalized with 18S values and are expressed as percentage of the IL-1ß 24-h response. *, A significant difference from similar values obtained in the presence of IL-1ß and INDO (Veh; Student’s t test).

 


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  		Figure 5. Antagonistic effect of SC-51322 on the 17-phenyl trinor PGE2-induced and IL-1ß-induced increases in IL-1ß mRNA levels in PLC. A, PLC were cultured for 24 h with IL-1ß (10 ng/ml), INDO (10 µM), and 17-phenyl-trinor-PGE2 (10 µM) in the presence or absence of SC-51322 (3 or 30 µM). B, PLC were cultured for 24 h with IL-1ß (10 ng/ml) in the presence or absence of SC-51322 (3 or 30 µM). Total RNAs were extracted, and cDNAs were amplified by real-time RT-PCR using primers and probes for IL-1ß. Results are means ± SEM from individual experiments (n, indicated in parentheses). Data are normalized with 18S values and are expressed as percentage of IL-1ß 24-h response. *, A significant difference from similar values obtained in the presence of IL-1ß and INDO [vehicle (Veh); Student’s t test].

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The findings of this study demonstrate that rat Leydig progenitors express FP, EP1, EP2, and EP4 prostaglandin receptors. Of these PG receptors, only EP1 and EP4 were detected in adult Leydig cells. A splicing variant of the EP3 receptor is expressed in both progenitors and adult Leydig cells. Four splice variants of the rat prostaglandin EP3 receptor have been identified to date (21). These modifications in RNA splicing result in multiple isoforms differing in the sequence of their intracellular C terminus. However, an antibody to the first extracellular, N-terminal domain of rat EP3, one that readily detects the EP3 receptor in murine MA-10 cells, fails to detect EP3 protein of any size in rat Leydig cells. Such findings may reflect either the relatively high abundance of this receptor in the immortalized Leydig tumor-derived cell line or the presence of a yet undetermined isoform of the rat EP3 receptor present in the mature rat Leydig cell and its progenitor. Sequencing of the EP3 PCR product revealed a sequence with a high level of similarity with EP3 receptor isoforms, suggesting that it may represent a fifth member of the rat EP3 receptor family. The EP3 receptor is known to have multiple splice variants in human (nine variants), cow (four variants), and mouse (three variants), and further studies will be needed to characterize the EP3 receptor isoform found in the rat Leydig cell.

IL-1ß treatment markedly decreases EP1 receptor levels, and INDO abrogates this down-regulation. In contrast, FP, EP2, and EP4 receptor levels modestly increase after IL-1ß treatment. Pharmacological experiments demonstrate that in progenitors the selective activation of FP and EP1 receptors increases IL-1ß expression. EP2, EP4, and EP3 agonists are without effect. Finally, SC-51322, an EP1 receptor selective antagonist, inhibits IL-1ß autoinduction similar to INDO.

Using whole cell lysates prepared from Leydig progenitors, specific anti-FP, EP1, EP2, and EP4 receptor antibodies detected only one distinct protein band. However, apparent mobilities for FP, EP2, and EP4 receptors (approximately 60, 90, and 80 kDa, respectively) were different from the theoretical molecular masses predicted from the amino acid sequence (41, 40, and 53 kDa, respectively; Refs. 22 and 23). In contrast, the apparent mobility for EP1 (~44 kDa) was similar to its theoretical molecular mass (43 kDa; Ref. 23). Such differences in kilodaltons were reported for EP receptors in human kidney and embryonic rat sensory neurons (18, 24). These authors proposed that the variation in size may reflect posttranslational modifications of the receptor protein such as glycosylation and/or phosphorylation. In embryonic rat sensory neurons, molecular masses for EP1, EP2, and EP4 receptors range from 63–67 kDa (18). A higher molecular mass (78 kDa) EP4 receptor was reported in the human kidney (24). Comparison of these data and the results in the present study suggest that the apparent molecular mass of the PG receptors varies with the cell type and/or developmental stage within the same species.

In part, FP and EP receptor expression in adult testis was previously indicated. EP2 mRNA was detected in the rat testis, and EP3 mRNA was shown in the human testis, albeit at a much lower abundance than in kidney, pancreas, or uterus (23, 25). In Leydig progenitors, our data provide evidence for the presence of EP2 but not EP3 receptor proteins. In adult mouse testis, FP receptor immunohistochemical staining was associated with a subpopulation of adult Leydig cells observed in the interstitium (26). Our results indicate that FP receptors are present in Leydig progenitors but not in adult Leydig cells of the rat testis. Additionally, progenitor FP receptor expression can be regulated by IL-1ß. The apparent differences between the two studies may reflect the methods used, species specificity, or the maturational state of the particular subpopulation observed in the adult mouse. To date, little is known about EP4 and EP1 expression in the testis. Although EP4 receptors are expressed in a variety of tissues such as thymus, ileum, lung, spleen, adrenal, and kidney tissues, EP1 receptors have a more limited range of tissue expression. EP1 receptors are highly expressed in the kidney and are expressed to a lesser extent in the gastric muscularis mucosae and adrenal tissue (27). Our data demonstrate that EP4 as well as EP1 receptors are expressed in progenitors and adult Leydig cells. The present study is the first report showing a distinct pattern of expression for the FP, EP1, EP2, EP3, and EP4 prostaglandin receptors during postnatal development and maturation of Leydig cells. Further study will be required to determine the functional significance of these developmentally differential patterns.

In female reproductive tissues, prostaglandins, especially PGE2 and PGF2{alpha}, are known to play an important role in the regulation of ovulation, luteinization, luteolysis, and parturition (2). A coordinated regulation of COX2, FP, and EP receptors by IL-1ß was shown in those reproductive tissues involved in these processes. In human granulosa cells, mRNAs coding for COX2 and the FP, EP2, and EP4 receptors are induced by IL-1ß, whereas COX2 and EP4 mRNAs are induced by this interleukin in human myometrium (28, 29, 30). Such regulation of the inducible COX2 enzyme, which catalyzes the rate-limiting step of the PG cascade, and of the PG receptors suggests that both PG synthesis and sensitivity may be under auto and/or paracrine control (28). Similarly, our studies showed that IL-1ß induces COX2 mRNA in Leydig progenitors (12). The present data indicate that the mRNA levels of PG receptors are affected by IL-1ß as well. Similar to the regulation observed in human granulosa cells, Leydig progenitor FP, EP2 and EP4 receptors increased after IL-1ß treatment. Furthermore, these data show that the level of the progenitor EP1 receptor is dramatically decreased by IL-1ß. The data indicate that these effects of IL-1ß involve endogenous PG production because INDO abrogates this down-regulation.

EP2 and/or EP4 receptors are most commonly associated with immunological modulation. PGE2 inhibits TNF{alpha} induction by zymosan or lipopolysaccharide in mouse macrophages and neutrophils, human monocytes or Kupffer cells—effects mediated by EP2 and/or EP4 receptors (31, 32, 33, 34, 35, 36). Similarly, these receptors are involved in the IL-6, IL-8, and IL-10 induction by PGE2 in a variety of cell types (32, 35, 37, 38). However, EP1 and/or EP3 receptors mediate PGE2 induction of IL-6 in mouse mast cells and an osteoblast-like cell line (39, 40). The present study provides evidence of EP1 involvement in the PGE2 induction of IL-1ß in Leydig progenitors. In fact, 17-phenyl trinor PGE2, the preferential EP1 > EP3 agonist, was more potent than sulprostone, the preferential EP3 > EP1 agonist, and PGE2 to induce IL-1ß (Fig. 3Go; Ref. 3). Furthermore, the EP1 antagonist SC-51322 (41) inhibits 17-phenyl trinor PGE2 induction of IL-1ß in PLC treated with IL + INDO as well as IL-1ß itself. None of the EP2/EP3/EP4 agonists tested in this study affected IL-1ß mRNA levels. Taken together, our studies demonstrate that EP1 receptor activation mediates exogenous as well as endogenous PGE2 induction of IL-1ß in PLC. These data are consistent with the observed stimulatory effect of a low dose (0.1 µM) of cloprostenol, a selective agonist for FP receptors (3), on IL-1ß mRNA levels in PLC treated with IL-1ß + INDO. FP and EP1 receptors are coupled to the same pathway, i.e. intracellular calcium mobilization. Although Noguchi et al. (42) have shown that PGF2{alpha} up-regulates IL-6 production in human gingival fibroblasts, the present study provides the first evidence for the involvement of FP receptors in the regulation of cytokine production. On the basis of the data herein, such a potential FP regulatory component may not be present after Leydig cell differentiation. Because a FP receptor-specific antagonist is not currently available, further investigation of FP receptor-regulated, endogenous PG-mediated induction of IL-1ß in PLC is not possible at this time. However, SC-51322 (30 µM; Fig. 5Go) and INDO have a similar (60%) inhibitory effect on IL-1ß autoinduction, suggesting that EP1 antagonism is enough to block the endogenous PG-mediated regulation of IL-1ß mRNA levels in PLC. At this level, the contribution of FP receptors may then be less important. This hypothesis is consistent with the 2-fold greater production of PGE2 than PGF2{alpha} observed after treatment of progenitors with IL-1ß (12).

In conclusion, our data demonstrate that FP and EP1 receptors mediate PGF2{alpha} and PGE2 induction of IL-1ß expression in a progenitor cell lineage in the testis. The results are consistent with an endogenous PG-regulated IL-1ß autoinduction mediated by EP1 receptors. Furthermore, these receptors are down-regulated by IL-1ß, findings suggestive of an autocrine feedback mechanism used by progenitors to protect themselves from a sustained deleterious production of the testicular cytokine IL-1ß.


   Acknowledgments
 
We express our appreciation for the expert primary cell preparations by Lyann Mitchell, excellent technical assistance by KeumSil Hwang, and editorial help by Jean Schweis.


   Footnotes
 
This research was funded by NIH Grant R01-HD-39024 (to P.L.M.). Access to the Cell Culture Core Facility of the Population Council was provided by National Institute of Child Health and Human Development/NIH support through a cooperative agreement (U54-HD-13541) as part of the Specialized Cooperative Centers Program in Reproduction Research.

Abbreviations: COX, Cyclooxygenase; DMSO, dimethylsulfoxide; EP receptors, specific receptors for PGE2; FP receptors, specific receptors for PGF2{alpha}; 3{alpha}-HSD, 3{alpha}-hydroxysteroid dehydrogenase; INDO, indomethacin; PG, prostaglandin(s); PLC, progenitor Leydig cells; RT, reverse transcription; SD, Sprague-Dawley.

Received August 20, 2002.

Accepted for publication January 6, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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