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Endocrinology Vol. 138, No. 1 237-247
Copyright © 1997 by The Endocrine Society

ARTICLES

Cloning of Guinea Pig Cyclooxygenase-2 and 15-Hydroxyprostaglandin Dehydrogenase Complementary Deoxyribonucleic Acids: Steroid-Modulated Gene Expression Correlates to Prostaglandin F2{alpha} Secretion in Cultured Endometrial Cells1

K. E. Bracken, W. Elger, I. Jantke, A. Nanninga and B. Gellersen

Institute for Hormone and Fertility Research, Division of Reproductive Sciences, University of Hamburg, 22529 Hamburg, Germany and Entec GmbH (W.E.), 07745 Jena, Germany

Address all correspondence and requests for reprints to: Birgit Gellersen, Institute for Hormone and Fertility Research, Division of Reproductive Sciences, Grandweg 64, 22529 Hamburg, Germany. E-mail: 100607.1557{at}compuserve.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prostaglandin F2{alpha} (PGF2{alpha}) secretion is lowest at midcycle and highest on day 15 at luteolysis in the cycling guinea pig uterus and is inversely related to serum progesterone levels. An increase in 17-ß estradiol (E2) occurs only towards the end of the cycle. To investigate the effect of steroids on the control of uterine PGF2{alpha} metabolism at the level of gene expression we established a primary cell culture model of day 15 cycling guinea pig endometrial cells. We cloned guinea pig cDNAs for cyclooxygenase 2 (COX-2), 15-hydroxyprostaglandin dehydrogenase (PGDH) that converts PGF2{alpha} to biologically inactive 13,14-dihydro-15-keto PGF2{alpha} (PGFM) and a fragment of cyclooxygenase-1 (COX-1). They were found to bear 87% and 90% homology at the amino acid level to their human counterparts for COX-2 and PGDH, respectively, retaining all functional sites. Purified epithelial and stromal cell subcultures were primed with medium containing either E2 or medroxyprogesterone acetate (MPA) for 24 h. They were then treated for a further 4 or 24 h either withdrawing the steroid, maintaining the priming steroid, or supplementing with both steroids, before harvesting conditioned media and RNA. Epithelial cells secreted 30-fold more PGF2{alpha} compared with stromal cells (e.g. 7.8 ± 0.7 vs. 0.26 ± 0.09 pg/ng DNA•24 h), and PGF2{alpha} secretion levels were approximately 15-fold higher than those of PGFM (e.g. 7.8 ± 0.7 vs. 0.45 ± 0.16 pg/ng DNA·24 h, for epithelial cells). COX-1 transcripts were low and unaffected by treatment in both cell types. COX-2 transcripts were more abundant in epithelial than stromal cells. Steroid-modulated, COX-2 dependent changes in PGF2{alpha} secretion were observed. The addition of MPA to E2 primed cells caused a decrease in PGF2{alpha} secretion and COX-2 messenger RNA levels after 4 h. Conversely, the addition of E2 to MPA primed epithelial cells led to an increase in PGF2{alpha} secretion and COX-2 messenger RNA levels after 4 and 24 h. The withdrawal of E2 caused a fall in PGF2{alpha} secretion and COX-2 transcripts after 24 h. In contrast, PGDH transcripts were more abundant in stromal than epithelial cells and were up-regulated by the addition of MPA to E2 primed cells. These in vitro observations are in keeping with the secretory profile seen in vivo in the cycling guinea pig uterus suggesting that 1) the fall of E2 and the coinciding rise in progesterone seen in the early cycle lead to a reduction in PGF2{alpha} levels; and 2) the rise of E2 in the late cycle on a progesterone primed uterus is the stimulus for an increase in uterine PGF2{alpha} production. Our findings suggest a differential role for uterine stroma and epithelium in vivo whereby the former acts to remove (via PGDH), and the latter to produce (via COX-2) biologically active prostaglandin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FOR THE ESTABLISHMENT and maintenance of pregnancy, uterine PGF2{alpha} production must be suppressed to conserve a quiescent uterus, and in some species to prevent luteal regression (1). The guinea pig who, among the nonprimates, bears the closest resemblance to human female reproductive physiology (2), has been used extensively as a model to study the control of uterine PGF2{alpha}. Progesterone and PGF2{alpha} levels in the cycling guinea pig uterus are inversely related, whereas the rise in PGF2{alpha} towards the end of the cycle coincides with the increase in 17ß-estradiol (E2) (3, 4). This indicates that steroids may play an important role in the control of PGF2{alpha}. Experiments in intact guinea pigs using progesterone agonists and antagonists suggest that progesterone alone stimulates uterine PGF2{alpha} production (5, 6). Experiments in vitro on uterine cells isolated from the guinea pig and other species suggest that E2 acting on a progesterone-primed endometrium is the stimulus for increased uterine PGF2{alpha} production (reviewed in Ref.7).

The enzyme responsible for a rate limiting step in PG synthesis exists as two isoforms, COX-1 and COX-2, that are encoded by separate genes. They perform a two-step conversion of arachidonic acid to PGH2 via PGG2. The three-dimensional protein structure for COX-1 has been elucidated (8), and that of COX-2 is presumed to be similar owing to a high amino acid homology. The enzyme has an EGF-like domain, aspirin acetylation site, an enzymatic domain with two distinct active sites for its peroxidase and cyclooxygenase activities, and a membrane binding motif locating it to either the endoplasmic reticulum or nuclear membrane (reviewed in Ref.9). COX-1 is constitutively expressed in most cell types and has been considered a housekeeping gene (10), although more recent evidence shows regulated expression in both the inflammatory and reproductive systems (11, 12). COX-2 is a dynamic isoform induced by specific stimuli in specific cell types (13). Unlike COX-1, COX-2 gene expression is regulated not only at the transcriptional level, e.g. by cAMP, NF-IL-6, NF{kappa}B and C/EBP (9), but also by multiple sequences in its 3' untranslated region (UTR) which are involved in destabilization of the messenger RNA (mRNA) (14, 15, 16).

In vivo, PGF2{alpha} is rapidly catabolized into its stable, biologically inactive metabolite 13,14-dihydro-15-keto PGF2{alpha} (PGFM). Plasma PGFM is often measured instead of PGF2{alpha} as the former is present at much higher levels allowing easier quantitation. The cytosolic enzyme responsible for this catalysis is the NAD+ dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) (17), which is present at high levels in the lung, liver, and placenta. In the feto-placental unit, PGDH has been implicated in the maintenance of pregnancy, as high PGDH activity in the chorion protects the myometrium from the high levels of PGF2{alpha} produced by the amnion (18, 19, 20). A number of correlations between PGDH enzyme activity and progesterone levels in human placental and uterine tissues have led to the suggestion that the enzyme is under steroidal control (21, 22, 23). It can be inferred therefore that the COX isoenzymes and PGDH in concert determine the level of biologically active prostaglandins.

The aim of this study was to establish a primary cell culture system of purified endometrial stromal and epithelial cells with which to investigate their respective contributions to the control of uterine PGF2{alpha} at the level of gene expression. We present the cloning of guinea pig PGDH and COX-2 cDNAs and evidence for their control of PGF2{alpha} levels in the different endometrial compartments in response to steroids.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Purification and maintenance of primary cell cultures
Virgin guinea pigs of the HSD:WIN Durukim Hartley strain (Harlan-Winkelmann GmbH, Borchan, Germany) were kept in climatized rooms under artificial light from 0600 h to 1800 h (23 ± 1 C) and provided with food (standard altromin-diet) ad libitum and tap water. They had a minimum body weight of 500 g and were examined daily for stage of the cycle, day 1 being the day of vaginal opening.

Uteri from day 15 of the nonpregnant cycle (two to three per experiment) were collected in chilled basic media (BM) consisting of a 1:1 mixture of Ham’s-F12:DMEM supplemented with 10% FCS, 4 mM glutamine, 20 mM HEPES, 100 U/ml penicillin, and 100 µg/ml streptomycin. Purified cultures of endometrial stromal and epithelial cells were obtained as follows: uteri were cut open mesometrially and the myometrium dissected off before the endometrium was minced finely and digested at 37 C in 0.25% collagenase (CLS 1; Worthington, NJ) and 10 U/ml DNAse I (Sigma, Deisenhofen, Germany) in Ham’s-F12:DMEM. After 1.5 h, an initial 5-min bench sedimentation of undigested tissue was carried out and the sediment submitted to a second round of digestion for 1 h. Epithelial glands and single stromal cells were then separated by differential centrifugation of the supernatant as previously described (24) using HBSS for washing. Fractions were further purified by selective attachment. Stromal cells were plated in BM on flasks precoated with bovine fibronectin (Sigma; 20 µg/ml in PBS for 15 min at 37 C) and allowed to adhere for 30 min, during which time stromal cells only attached. The media containing contaminating epithelial cells was discarded. Epithelial cells were incubated in BM containing only 5% FCS for 30 min to remove adhering stromal cells before plating on flasks which had been coated with 50% FCS in Ham’s-F12:DMEM (1 h, 24 C).

Cultures were grown in BM supplemented with 10% or 5% FCS for stromal and epithelial cells, respectively, for 5–6 days until reaching confluency. Next they were submitted to differential trypsinization and attachment for further purification and subculture. Stromal cells were washed in PBS, trypsinized for 3–5 min in 1 x trypsin/EDTA (Sigma), and plated onto fibronectin-coated multiwell dishes (2 x 104 cells/ml). Epithelial cells were washed twice in PBS and selectively trypsinized with a 1:5 dilution of trypsin/EDTA, for 2 min or until contaminating stromal cells only were removed. After a fast wash in PBS, fresh 1 x trypsin/EDTA solution removed the epithelial cells for plating (2 x 104 cells/ml) onto dishes coated with 50% dextran coated charcoal-stripped FCS (FCSDCC).

Every subculture was examined by triple immunofluorescent staining to ensure purity. A monoclonal mouse antivimentin primary antibody (M725; Dako, Hamburg, Germany) and an antimouse dichlorotrianzinyl amino fluorescein conjugated secondary antibody (a stable fluorescein derivative; Dianova, Hamburg, Germany) were used for green fluorescent staining of stromal cells. A wide spectrum rabbit polyclonal anticytokeratin primary antibody (Z622; Dako), and an antirabbit Cy3 conjugated secondary antibody (a stable rhodamine derivative; Dianova) were employed for red fluorescent staining of epithelial cells. Cells were finally subjected to nuclear counterstaining (visualized blue) with DAPI (4', 6-diamino-2-phenylindole; Sigma).

All plastic materials were from Nunc (Roskilde, Denmark) except for 12-well-plates (Costar, Wallisellen, Sweden), and cell culture grade solutions were purchased from GIBCO-BRL (Eggenstein, Germany) unless otherwise stated.

Cell culture stimulation regime
Subcultures were placed in 24-well plates for immunocytochemistry, 12-well plates for harvesting conditioned media and DNA, and 6-well plates for harvesting RNA. After growth to 80% confluency all subcultures were primed for 24 h with stimulation media (SM) consisting of phenol red-free BM supplemented with 10% FCSDCC, 7 µg/ml insulin and either E2 (1 x 10-9 M; Sigma) or medroxyprogesterone acetate (MPA; 2.5 x 10-7 M; Sigma). After priming, SM was added for 4 h or 24 h, containing no steroid (withdrawal), the priming steroid alone (maintenance), both steroids, or E2 plus EGF (100 ng/ml; Boehringer Mannheim, Mannheim, Germany). All experiments were completed within 2 weeks from day one of culture. After harvesting, conditioned media were aliquoted and stored at -20 C until analysis of PGF2{alpha} and PGFM levels by ELISA. DNA was measured from quadruplicate wells (taken up in 500 µl buffer) by capillary adapted fluorometry following the manufacturer’s instructions (TKO-100 minifluorometer; Hoefer Scientific Instruments, San Francisco, CA). Secretion values were normalized to DNA content and statistical comparisons were performed by one-tailed heteroscedastic t test. RNA was extracted with RNA clean (AGS, Heidelberg, Germany) using 3–4 wells per treatment for Northern blot analysis and RT-PCR.

Prostaglandin ELISAs
We developed nonradioactive competitive immunoassays for the detection of PGF2{alpha} and PGFM in conditioned media containing 10% FCSDCC. Biotinylated tracers were prepared by coupling D-biotinoyl-1,8-diamino-3,6-dioxaoctan (Boehringer Mannheim) to the carboxyl group of the PG (PGF2{alpha}; Cayman Chemical, Ann Arbor, MI) (PGFM; Sigma) using the mixed anhydride procedure (25). Biotinylated tracer (50 µl) was added to each well of a flat bottom 96-well plate (Minisorb; Nunc) at a concentration of 14 nM for PGF2{alpha}, or 2 nM for PGFM. Standard or sample (100 µl/well), prediluted where necessary in unconditioned SM from the respective cell culture experiment, were then added to duplicate wells. Rabbit primary antibody (50 µl/well) was added to all wells except the nonspecific binding wells, which received ELISA buffer (see below) alone. The primary antibodies were: rabbit anti-PGF2{alpha}/BSA antibody no. 57 (a gift from Dr. R. W. Kelly, MRC, Edinburgh, UK) used at a 1:500,000 dilution, and a commercially available rabbit anti-PGFM antibody (no. PG003; S. Klinger, St. Albans, UK) used at a 1:250,000 dilution (recommended dilution for RIA, 1:200). Finally lids with 96 pins corresponding to the 96 wells (pin-plates; Maxisorb, Nunc) were placed onto the 96-well plates and incubated overnight shaking at 4 C in a dark and humid chamber. The pin-plates had been precoated with a goat antirabbit secondary antibody (1 µg/well; Scantibodies, Santee, CA) as previously described (26) and stored at -20 C until use. The pin-plates were then removed from the well plates, shaken dry, and incubated in a fresh 96-well plate containing 200 µl/well of 150 ng/ml horseradish peroxidase-streptavidin solution (Vector Labs, Burlingame, CA), for 15 min at 4 C (dark and humid). This was followed by two washes in a tray of ELISA buffer (4 C), and one wash in 0.05% Tween-20 in H2O (24 C), each for 15 sec under gentle agitation. The pin-plates were then incubated in fresh 96-well-plates containing 250 µl/well of enzyme substrate solution at 24 C (in a dark, humid chamber). The reaction was stopped after 40 min by the addition of 2 M H2SO4 (50 µl/well), which also led to a color change from blue to yellow. The OD450 of each plate was analyzed using a plate reader and EasyFIT program from SLT Labinstruments (Crailsheim, Germany). All dilutions unless otherwise stated were in ELISA buffer (0.15 M NaCl, 0.04 M NaH2PO4, 0.2% BSA fraction IV [Sigma], pH 7.2 and 0.025% Thimerosal). The enzyme substrate buffer was 0.1 M acetic acid solution adjusted to pH 5.5 with citric acid to which H2O2 (0.004%) and 3,3',5,5'-tetramethylbenzidine (0.01%) were freshly added.

Validation analysis of the assays was carried out using six independent ELISAs on different days, carrying triplicate standards and samples in six parallel wells. The following characteristics were determined: sensitivities of the assays, defined as the smallest standard which was different by 3 SD from the zero standard, were: PGF2{alpha},11 pg/ml; PGFM, 37 pg/ml. The mid ranges of the standard curves, defined as the PG concentrations giving 50% specific binding (B/Bo), were: PGF2{alpha}, 70 pg/ml; PGFM, 300 pg/ml. The limits of quantitation, defined as the smallest measurable concentrations where the coefficient of variation (CV) was less than 15%, were: PGF2{alpha}, 14.8 pg/ml; PGFM, 50 pg/ml. The reproducibility of the assays (interassay CVs between six independent ELISAs), was: PGF2{alpha}; 8.22% for 720 pg/ml, 9.42% for 360 pg/ml, 9.58% for 90 pg/ml, and 11.87% for 22.5 pg/ml; PGFM; 10.54% for 2600 pg/ml, 13.42% for 650 pg/ml, and 14.83% for 162.5 pg/ml. The precision of the assays (intraassay CVs within one individual ELISA), was: PGF2{alpha}; 7.70% for 720 pg/ml, 8.88% for 360 pg/ml, 1.91% for 90 pg/ml and 4.63% for 22.5 pg/ml; PGFM; 9.21% for 2600 pg/ml, 8.62% for 650 pg/ml, 13.32% for 162.5 pg/ml. Cross-reactivities in the PGF2{alpha} ELISA were: PGFM, 0.22%; PGE2, 0.55%. Other cross-reactivities for the PGF2{alpha} antibody over 0.2% are: PGF1{alpha}, 7.2%; PGF3{alpha}, 2.9%; PGF2ß, 3.5%; 6-oxoPGF1{alpha}, 1.05% (27). Cross-reactivities in the PGFM ELISA were: PGF2{alpha}, 0.027%; PGE2, <0.001%. Other cross-reactivities for the PGFM antibody over 0.01% are: 15-ketoPGF1{alpha}, 4.2%; 13,14-dihydroPGF2{alpha}, 2.2% (S. Klinger). Recovery analysis by serially diluting conditioned media samples with initial PG concentrations at least at the upper limit of the standard curves (duplicate wells from six independent experiments; means ± SE) yielded: PGF2{alpha}, 96 ± 14.4%; PGFM, 95.4 ± 7%.

PGF2{alpha} and PGFM assays for conditioned media from individual experiments were carried out on the same day, keeping treatment groups for specific time points together on a plate. Each ELISA plate carried its own standard curve with a range of 7.4–800 pg/ml for PGF2{alpha} and 27–3000 pg/ml for PGFM. Values below 20 pg/ml and 50 pg/ml for PGF2{alpha} and PGFM, respectively, were considered under the confidence limits of detection.

Isolation of guinea pig specific cDNA probes for Northern blot analysis
Fragments of guinea pig COX-1, COX-2, and PGDH cDNAs were cloned from day 50 placenta (COX-1), or a culture of mixed endometrial cells stimulated with E2 plus EGF (COX-2, PGDH), by RT-PCR. cDNA was synthesized from RNA using Superscript RNase H- reverse transcriptase (GIBCO-BRL) and oligodeoxythymidine primers. PCR was performed with 1.5% of the resulting cDNA in the presence of 0.2 µM of 3'- and 5'-primers, 0.2 mM dNTPs, and 1 U of Pfu polymerase (Stratagene, Heidelberg, Germany) in a final volume of 50 µl. A touchdown program using annealing temperatures from 61 to 51 C was used in a heated lid thermocycler (Hybaid, Teddington, UK). With respect to the COX isoenzymes, primers were based upon the human (28, 29), rat (30), mouse (31, 32), and ovine (33) sequences. Regions were chosen which have low homology between isoforms but are highly conserved between species. The primers used were (numbers in brackets indicate the nucleotide position relative to the human cDNA): COX-1-sense, 5'-GTGTGACCTGCTGAAGGCTGAGCAC-3' (944 to 968). COX-1-antisense, 5'-CTTGCGGTACTCATTGAAGGGCTGC-3' (1388 to 1412). COX-2-sense, 5'-GACCAGAGCAGGCAGATGAAATAC-3' (1412 to 1435). COX-2-antisense, 5'-CTGTGGGATTGATATCATCTAGTC-3' (1813 to 1836). The PGDH primers were designed upon the human placental sequence (34), yielding almost the full protein coding region of 714 bp: PGDH-sense, 5'-CGTGAACGGCAAAGTGGCGCTGGTG-3' (35 to 50). PGDH-antisense, 5'-GCTAAAGATGACATATTGATAATG-3' (765 to 788).

Positive clones were obtained using pCR-Script SK(+) cloning kit (Stratagene), and sequencing was carried out with Sequenase T7 DNA polymerase (United States Biochemical, Cleveland, OH). Northern analysis was performed as previously described (35) using 50 µg total RNA/lane extracted from tissue using the guanidine thiocyanate-CsCl method (36), or 20–25 µg of total RNA/lane for cell culture samples. Control hybridizations were performed with a 2-kilobase pair (kb) insert of chicken ß-actin cDNA (35). COX-2 and PGDH transcript abundance from two to six independent experiments was analyzed by densitometric scanning of autoradiograms and normalized to ß-actin mRNA abundance. Sequence homologies to other species were determined using the Martinez Needleman-Wunsch and Lipman-Pearson methods (ALIGN; DNASTAR, Inc., Madison, WI) for nucleotide and amino acid sequences, respectively.

COX-2 cloning strategy
A unidirectional cDNA library was prepared from guinea pig uteri (day 15 of the nonpregnant cycle) using a Uni ZAP-cDNA synthesis kit (Stratagene) yielding one million independent clones. Screening was performed using the 375 bp guinea pig COX-2 fragment (see above) labeled by PCR in the presence of digoxygenin-11-dUTP (0.14 nmol/µl; Boehringer Mannheim). Hybridization was carried out at 60 C with 5 ng/ml of labeled probe (37) and detected with DIG luminescent detection kit for nucleic acids (Boehringer Mannheim). One clone of 865 bp in length was identified. The published COX-2 sequences for other species, and Northern analysis of guinea pig tissues indicated that the full length sequence encompassed approximately 4 kb.

Various PCR strategies were adopted to obtain the remaining sequence. Specifically primed cDNAs were synthesized using total RNA from cultured E2 stimulated epithelial cells as the template: 1) A sense primer designed upon the 5' end of the human protein coding region (COX-2E) paired with a downstream guinea pig specific primer (COX-2D) yielded a 1.6 kb PCR product, using a COX-2D primed cDNA, Taq polymerase (Promega, Madison, WI) and a touchdown program with annealing temperatures of 62 to 50 C (48 cycles in total) and 3 min extension time. The final PCR products were cloned into pGEM-T (Promega). 2) Part of the presumed 5'-UTR and the missing N-terminal protein coding region were obtained using a 5' rapid amplification of cDNA ends (RACE) inverse PCR method (38). For this the first strand cDNA synthesis was primed with COX-2P, a guinea pig specific oligonucleotide. Following second strand synthesis the double stranded products were circularized by self-ligation. The unknown sequence was then amplified by PCR using the guinea pig specific primers COX-2R and COX-2S, Pfu polymerase and 1 min extension time, and cloned into pCR-Script. 3) The 3' region was partially amplified using a 3' RACE method and cloned into pGEM-T. Primary strand cDNA synthesis was primed with a 35-mer anchor primer, 5'-GACTCGAGTCGACATCG[T17]-3'. PCR was performed using the Expand long template PCR system (Boehringer Mannheim), with guinea pig specific COX-2F as the 5' primer, and the anchor primer minus [T17] as the 3' primer.

The primer sequences were (numbers in brackets indicate the nucleotide position relative to the human cDNA): COX-2P; (antisense) 5'-GCGCAGGAAGGGAATGTTATTG-3' (361 to 382). COX-2R; (antisense) 5'-GGTACAGTTTTCACCGTAATAGGC-3' (239 to 261). COX-2S; (sense) 5'-CCCAACACGGTGCACTATATAC-3' (308 to 329). COX-2E; (sense) 5'-ATGCAGCAAATCCTTGCTGTTC-3' (143 to 165). COX-2D; (antisense) 5'-CGTTATTGCAGATGAGAGACTGGATGG-3' (1743 to 1769). COX-2F; (sense) 5'-CCGCAAACGCTTCTTGATGAAACCG-3' (1453 to 1477).

PGDH cloning strategy
To obtain the full protein coding region of guinea pig PGDH, we adopted a PCR strategy using the unidirectional guinea pig uterus cDNA library. Four guinea pig specific primers were designed upon the sequence inside the 5' (PGDH-5 and PGDH-10; antisense) and 3' (PGDH-7 and PGDH-11; sense) ends of the 714-bp fragment obtained by RT-PCR (see above). PCR was carried out using primers flanking the polylinker of the pBluescript SK(+) vector (pBSK) of the cDNA library paired with PGDH specific primers (PGDH-5 or PGDH-7), 2 µl of phage suspension as a template and Tth DNA polymerase (Epicenter, Madison, WI). This was followed by nested PCR using pBSK-specific primers paired with PGDH-10 or PGDH-11. The 5' end was amplified with Pfu polymerase, the 3' end with Taq polymerase, and products were cloned into pCR-Script or pGEM-T, respectively. For all PCRs a hot start touchdown program with annealing temperatures of 55 C to 45 C and a 1-min extension time were used. The primer sequences were as follows (numbers in brackets indicate the nucleotide position relative to the human PGDH cDNA): PGDH-5; 5'-CATTATTCACTCCAGCATTATTGAC-3' (265 to 289). PGDH-10; 5'-CTTCAAGATTCGAGTCCACCC-3' (103 to 122). PGDH-7; 5'-GCCTGTTGCACAACAGCCTG-3' (429 to 449). PGDH-11; 5'-CACTCATTGAAGATGATGATTTG-3' (692 to 714).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning and sequence analysis of guinea pig COX-2 cDNA
We cloned the full protein coding region and part of the 3' UTR of the guinea pig COX-2 cDNA. We initially screened a cDNA library from a day 15 cycling guinea pig uterus, at which point COX-2 expression was expected to be high. We isolated one clone of 865 bp located at the 3' end of the protein coding region (Fig. 1Go). Comparison with the published sequences for COX-2 in other species, and the detection of a 4-kb transcript in guinea pig tissue (see Fig. 4Go) indicated that this clone encompassed only part of the COX-2 transcript. We therefore adopted various PCR strategies to isolate the remaining COX-2 sequence (Fig. 1Go) using RNA prepared from cultured epithelial cells which showed high levels of COX-2 mRNA upon Northern hybridization.



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  		Figure 1. Scheme of cloning strategy used for the guinea pig COX-2 cDNA. The clones obtained and the methods employed to obtain them are indicated, with the primers used for PCR boxed at each end of the respective clones. The thick solid line at the top represents the coding region and the thick broken lines indicate the 5' and 3' UTRs. The double-stranded circle clarifies the 5' RACE inverse PCR strategy; primer 2P was used for primary strand synthesis, 2S and 2R for PCR amplification, whereas the inner broken curve represents the previously unknown portion.

 


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  		Figure 4. Northern hybridization of guinea pig tissues for COX-1, COX-2, and PGDH. A, Northern blot carrying 50 µg/lane total RNA from the ovary of a day 12 pregnant guinea pig (lane 1), and day 50 placental tissue (lane 2) was hybridized with COX-1, COX-2, and PGDH guinea pig specific probes. B, Northern blot of cultured epithelial cells grown in the presence of E2 and MPA, hybridized with COX-2, showing an additional transcript compared to the single one seen in tissue. Sizes of the transcripts are indicated on the right.

 
The coding region of guinea pig COX-2 cDNA (Fig. 2Go) shows high homology to its human counterpart (29) at the nucleotide and amino acid level (82.6% and 87.1%, respectively). Homologies to other species are: rat 88.7% (30), mouse 88.4% (32), and chicken 79.8% (39). The ATG translational start site is presumably the same as that of the human as the surrounding nucleotides in both species are almost conserved and comply with the Kozak sequence (40). The transmembrane domain is fully conserved compared with the human. Other important functional sites such as those for heme coordination, aspirin acetylation, putative glycosylation, and active site tyrosine are also conserved. The 3' UTR is dissimilar from that of the human. However, it also contains multiple ATTTA sequences characteristic of the COX-2 3'-UTR in other species (29, 30, 31, 32, 39) associated with decreased stability of the mRNA. Two putative polyadenylation sites were also identified in the form of ATTAAA and AATAAA.



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  		Figure 2. Nucleotide and deduced amino acid sequences of the guinea pig COX-2 cDNA. Nucleotides and amino acids are numbered relative to the first ATG initiator codon. Doubly underlined are the putative membrane spanning domain in the coding region and polyadenylation sites in the 3' UTR. The active site tyrosine is marked with an arrow, the aspirin acetylation site is indicated with an open triangle and the heme coordination sites with a filled triangle.Instability associated sequences are underlined, and putative glycosylation sites are underlined and marked with an asterisk.

 
Cloning and sequence analysis of guinea pig PGDH cDNA
We cloned the guinea pig PGDH cDNA by a combination of PCR strategies. We initially obtained almost all of the protein coding region using RT-PCR and primers based on the human sequence within the coding region. To obtain the remaining 5' and 3' translated and untranslated regions, we carried out PCR on the guinea pig cDNA library, as Northern blot hybridization of RNA from the same tissue showed a high level of PGDH expression (data not shown). Guinea pig specific PGDH primers were used paired with the primers in the respective flanking sequence of the pBSK vector of the library. We cloned approximately 80 bp of 5' UTR and 1 kb of 3' UTR, which included a poly(A) tail preceded by an ATTAAA polyadenylation signal about 40 bp upstream (data not shown). The coding region and amino acid sequence share an 88.4% and 90% homology, respectively, with the human PGDH (Fig. 3Go) with the Kozak sequence being conserved in the same context as in the human (34). The amino acid sequence is particularly highly conserved within the region, which is characteristic of the family of short-chain alcohol dehydrogenase enzymes (human position 149–164) with only a single amino acid substitution from isoleucine to valine at site 158 (41). Also within this region, the strictly conserved tyrosine 151, and the highly conserved lysine 155 are present which have been shown to be critical for enzymatic activity (42). In addition, all amino acid residues thought to be involved in formation of the catalytic site are conserved (43). Human PGDH is reported to have heterozygous proteins owing to an amino acid substitution at position 217 from tyrosine to cystein. We observed the tyrosine form in the guinea pig. The carboxy terminus bears relatively low homology to the human counterpart and has two consecutive stop codons.



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  		Figure 3. PGDH amino acid sequence homology between guinea pigs and human. Numbers on the right indicate the presumed guinea pig (upper sequence) amino acid position relative to that of the human (lower sequence) (55). Vertical bars join identical amino acids, two dots or one dot join highly or less highly related residues, and unjoined residues have no similarity. Stop codons are shown as filled diamonds. The underlined region indicates the amino acids which are highly conserved between all short-chain alcohol dehydrogenases, and the only deviation between the human and guinea pig is marked with a cross. The strictly conserved tyrosine 151 and highly conserved lysine 155 within this region are labeled with open triangles. Amino acids presumed to be located at the catalytic site are indicated by open and filled triangles. Tyrosine 217, which in the human can be substituted for a cystein, is marked with an arrow.

 
Detection of COX-1, COX-2, and PGDH transcripts in guinea pig tissues
Fragments of guinea pig COX-1, COX-2, and PGDH cDNAs were prepared by RT-PCR for use as probes in Northern blot analyses of tissues (Fig. 4Go). The COX-1 fragment of 470 bp with a homology of 87% to its human counterpart (28) detected one transcript of 2.8 kb in ovary of day 12 and placenta of day 50 of pregnancy. A second transcript of 5.2 kb reported in the human was not observed (44). The COX-2 fragment detected a transcript of approximately 4 kb in guinea pig placenta but not ovary. PGDH cDNA hybridized to one transcript of 2.4 kb in guinea pig ovary but not placenta. The 3.4 kb PGDH transcript previously reported in human placenta was not detected (34). An additional COX-2 transcript of approximately 2.6 kb was occasionally observed in epithelial cell preparations where the prominent 4 kb COX-2 species was abundant.

Steroidal regulation of PGF2{alpha} and PGFM secretion
To study steroidal regulation of endometrial PGF2{alpha} production, we established a primary cell culture model of purified endometrial stromal and epithelial cells. Subculture purities were almost 100% for stromal and between 80% and 95% for epithelial cells in different tissue preparations (Fig. 5Go). Secretion of PGF2{alpha} and PGFM by these stromal and epithelial cells was measured in conditioned media using the ELISAs described above. Epithelial cells secreted on average 30-fold more PGF2{alpha} than stromal cells (e.g. 7.8 ± 0.7 vs. 0.26 ± 0.9 pg•ng DNA•24 h, respectively, within one representative experiment in the presence of E2). PGF2{alpha} secretion in epithelial cells was markedly higher than that of PGFM (e.g. 7.8 ± 0.7 vs. 0.45 ± 0.16 pg/ng DNA·24 h in the presence of E2 within the same representative experiment). The stimulation strategy was chosen to mimic the steroidal environment seen in vivo in the nonpregnant guinea pig uterus: 1) During the early cycle when PGF2{alpha} levels are low and still decreasing, the uterus is E2 primed and progesterone levels are rising. We mimicked this in vitro by priming the epithelial and stromal primary subcultures with E2 (24 h) and then either withdrew the E2, maintained it, or combined it with MPA, for 4 h or 24 h. As a positive control for the stimulation of COX-2 mRNA expression (13) and PGF2{alpha} secretion an additional treatment of E2 with EGF was included. 2) During the late cycle when uterine PGF2{alpha} levels are increasing, progesterone levels are falling while E2 is rising. We mimicked this in vitro by exposing the same preparation of cells to the reverse treatment of scenario (1); cells were primed with MPA (24 h), and then we either withdrew the MPA, maintained it, or combined it with E2 added, for 4 h or 24 h.



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  		Figure 5. Immunofluorescent staining of purified epithelial and stromal primary cell subcultures. Epithelial cells (A) were stained with anticytokeratin and stromal cells (B) with antivimentin antibody (50x).

 
Prostaglandin concentrations were normalized to DNA and calculated as a percentage of their respective controls, where the control was the maintenance of treatment with the priming steroid alone. Then results from four to eight individual experiments were pooled. This was necessary as absolute secretion values varied between individual tissue preparations. Steroidal modulation of PG secretion was observed (Fig. 6Go). The addition of MPA to E2 primed epithelial cells caused a decrease in PGF2{alpha} and PGFM secretion of 21% and 18%, respectively, after 4 h. After 24 h, this decrease was 12.2% for PGF2{alpha}. Conversely, the addition of E2 to MPA primed epithelial cells caused an increase in PGF2{alpha} and PGFM secretion that reached 75% and 59% of controls, respectively, after 24 h, and which became detectable for PGF2{alpha} secretion after 4 h. Withdrawal of E2 in epithelial cells led to a decrease in PGF2{alpha} and PGFM secretion of 33% and 36%, respectively, after 24 h. In epithelial cells, treatment with EGF transiently stimulated PGF2{alpha} secretion after 4 h to 161% of the control.



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  		Figure 6. Secretion of PGF2{alpha} and PGFM by cultured epithelial cells. Epithelial cell cultures were subjected to E2 (filled bars) or MPA (hatched bars) priming as detailed in Materials and Methods. Shown are the effects of withdrawal, maintenance of the priming steroid, or addition of both steroids on PGF2{alpha} (upper panel) and PGFM (lower panel) secretion. Treatment with E2 plus EGF is also shown. Conditioned media and DNA were harvested from quadruplicate wells after 4 h (left panel) or 24 h (right panel) of treatment. PGF2{alpha} and PGFM concentrations were normalized to the DNA content of the respective wells. Data are expressed as a percentage of the respective control (means ± SEM, with n = four to eight independent experiments), where the control is with maintenance of the priming steroid alone. ***, P <= 0.001; **, P <= 0.01; *, P <= 0.05, compared with respective controls.

 
In stromal cells, addition of EGF was the only treatment that resulted in PG secretion above the detection limit of our assays after 4 h. After 24 h, this rise amounted to approximately 1000% and 278% increase over the control for PGF2{alpha} and PGFM secretion, respectively (data not shown).

Regulation of COX-2 and PGDH mRNA abundance
RNA isolated from epithelial and stromal cells treated as above for 4 and 24 h was subjected to Northern blot hybridization with COX-1, COX-2, and PGDH probes (Fig. 7Go A, B). COX-1 transcripts of 2.8 kb were low and unaffected by treatment or nondetectable in both cell types (data not show). The prominent COX-2 and PGDH messages were of the same size as those observed in tissue (see Fig. 4Go). All hybridizations were normalized against ß-actin (Fig. 7Go C, D). It has to be noted that actin mRNA levels in stromal cells differed between the E2 and MPA primed sets.



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  		Figure 7. Northern blot analysis of cultured endometrial cells. RNA was harvested from epithelial cells (A) and stromal cells (B) as outlined in Fig. 6Go after 4 h (left panel) or 24 h (right panel). Northern blots carrying 25 µg total RNA/lane were hybridized with COX-2 and PGDH cDNAs simultaneously. Subsequent hybridization with ß-actin was performed as a loading control. Densitometric scanning was performed on epithelial cell autoradiographs for COX-2/ß-actin (C), and on stromal cell autoradiographs for PGDH/ß-actin mRNA levels (D), after 24 h treatment. Filled bars represent E2 primed cells and hatched bars MPA primed cells. Data are expressed as a percentage of the respective control where the control is with maintenance of the priming steroid alone (means ± SEM, with n = four to six independent experiments except n = two for MPA primed stromal cells (D, right panel), where no statistical analysis was performed); **, P <= 0.01; *, P <= 0.05, compared with respective controls.

 
COX-2 mRNA was more abundant in epithelial than stromal cells and in the latter was almost undetectable after 24 h treatment. Conversely, PGDH transcripts were more abundant in stromal than epithelial cells within a specific tissue preparation. COX-2 mRNA levels increased in the presence of EGF, used as a positive control, in both cell types (results not shown). Steroid-modulated expression of COX-2 and PGDH was observed. Comparisons were made to the respective controls, where the control was with maintenance of the priming steroid alone. The addition of MPA to E2 primed epithelial cells caused a down-regulation of COX-2 mRNA levels, whereas the addition of E2 to MPA primed epithelial cells caused an up-regulation of COX-2 mRNA levels after 4 h (densitometry not shown) and 24 h. Withdrawal of E2 from epithelial cells led to a decrease in COX-2 mRNA levels. PGDH expression was significantly elevated in response to MPA in E2 primed stromal cells after 24 h. An increase in PGDH expression with the addition of E2 to MPA primed stromal cells was also observed, but owing to a reduced number of Northern blots for this particular treatment the results were not subjected to statistical analysis (Fig. 7Go, C and D).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cloning and sequencing of guinea pig COX-2 cDNA revealed a high homology to the COX-2 cDNA of other species. All functionally important amino acids of the enzyme are conserved (29, 30, 31). These include: the active site tyrosine and heme coordination sites required for enzymatic activity, the RSTEL carboxyl terminus thought to be the signal required for insertion into the endoplasmic or nuclear membrane, the putative transmembrane domain, number, and spatial conservation of the putative glycosylation sites and the aspirin acetylation site. We initially isolated a fragment of the COX-2 cDNA from a guinea pig uterine cDNA library. Sequence comparison, the lack of a polyadenylation signal, and tissue Northern blot analysis indicated that only part of the 3' UTR had been obtained terminating at position 2383 compared with the human, of an estimated 4 kb total length. 3' RACE methods procured additional 3' UTR sequence. The 3' UTR carries two alternative polyadenylation signals, and it also has an abundance of ATTTA sequences associated with decreased stability of the mRNA (14, 15). Both of these structural features could explain the shorter clones isolated. Degradation of the 3' end may have caused the oligo deoxythymidine primer used for the first strand cDNA synthesis to anneal to an internal adenosine rich region instead of absent poly(A) tail. Alternatively, the initial mRNA could have been a product of alternative polyadenylation corresponding to shorter transcripts such as those seen in cultured epithelial cell mRNAs.

The nucleotide and amino acid sequence for guinea pig PGDH is highly homologous to its human counterpart (34), the only other species published to date. We detected a transcript of approximately 2.4 kb in size, which probably corresponds to the 2.0-kb message reported in human placenta, whereas the alternative species of 3.4 kb was absent from both tissue and cell culture RNA preparations (45). All of the amino acid residues thought to be important in the formation of the catalytic site are conserved (43) as are tyrosine 151 and lysine 155 which, by site directed mutagenesis, have been shown to be necessary for human PGDH catalytic function (42). The sequence rendering PGDH a member of the short-chain alcohol dehydrogenase family is identical to the human except for one amino acid substitution (41). It is within this region that tyrosine 151 is strictly conserved and lysine 155 highly conserved throughout all members of this enzyme family including guinea pig PGDH. We initially attempted to clone PGDH using primers based on the human 5' and 3' ends of the protein coding region but were unsuccessful. This is explained by the relative lack of homology in the 3' end between the two species.

Human endometrial epithelial cells have previously been shown to contain high levels of COX protein in the secretory phase (46) and to be responsible for most of the endometrial PGF2{alpha} production (27, 47). Conversely, endometrial stromal cells produce more PGE2 in a COX-2 dependent manner (48). We show here the first evidence supporting a direct correlation of COX-2 mRNA levels to PGF2{alpha} secretion by uterine cells in response to steroids: 1) epithelial cells secreted more PGF2{alpha} than stromal cells and had distinctly higher levels of COX-2 mRNA; 2) withdrawal of E2 from epithelial cells led to a decrease in both PGF2{alpha} secretion and COX-2 mRNA levels after 24 h; 3) addition of MPA to E2 primed epithelial cells caused a decrease in both PGF2{alpha} secretion and COX-2 mRNA levels; 4) the addition of E2 to MPA primed cells markedly increased PGF2{alpha} secretion and COX-2 mRNA abundance. The time required for these changes in secretion and COX-2 mRNA levels to occur further substantiates a correlation between the two, e.g. the increased secretion with the addition of E2 to MPA primed cells was apparent after 4 h, within which time span COX-2 mRNA was already distinctly elevated. In conclusion, these data indicate that an increase in PGF2{alpha} production is, at least in part, COX-2 dependent and that both the past and present steroidal milieus contribute towards the control of COX-2 expression.

Although COX-2 mRNA expression was relatively rapidly induced (within 4 h), it is not known if this is due to a transcriptional effect or stabilization of the message. No steroid-responsive elements have as yet been identified in the COX-2 promoter (9), whereas multiple instability related regions are present in the 3' UTR. COX-2 expression has been shown to be elevated by hCG in human endometrial cells in culture by an increase in stability of the transcripts (49).

COX-1 appears to play no significant part in this system, as levels were unaffected and low or undetectable under all treatments. However, as mRNA was not harvested before 4 h of treatment, we cannot rule out a very early role for COX-1. Studies on knockout mice have shown that COX-1 is mainly involved in early inflammatory responses. Conversely, the relatively delayed induction of COX-2 is more important in the secondary embellishment of both inflammatory and reproductive events (50, 51, 52, 53).

The degree of stimulation in our defined in vitro system was moderate compared with the dramatic changes seen in the intact uterus. This may indicate that in vivo the steroids are acting in a permissive manner to allow and/or stimulate other cells or factors (which were not present in our culture system), to modulate COX-2 expression and PGF2{alpha} secretion. The stromal cells expressed very low levels of COX-2 mRNA and secreted approximately 30-fold less PGF2{alpha} than epithelial cells. This was not due to restricted substrate availability because the addition of EGF caused a large increase in PGF2{alpha} secretion along with an upregulation in COX-2 mRNA expression. The reduced response of stromal cells to progesterone compared to epithelial cells was not due to lack of progesterone receptor (PR) expression; semiquantitative RT-PCR for PR performed on cDNA prepared from a representative cell culture experiment showed similar expression in both cell types under all treatments (data not shown).

Immunohistochemical studies and enzymatic assays in human uterine tissues of the cycle and early pregnancy suggested a stimulatory effect of progesterone on PGDH (21, 54). In the human endometrium, PGDH protein levels are higher in secretory than in proliferative endometrium (22), and treatment with antiprogestins leads to a reduction of PG catabolism. We demonstrate by Northern analysis of purified endometrial cell populations that it is the stromal compartment that is responsible for the majority of PGDH expression. In addition, we show progesterone mediated regulation of PGDH expression in this defined system. PGDH mRNA was significantly increased in stromal cells by the addition of MPA to E2-primed cells.

The role of progesterone in the control of uterine PGF2{alpha} production has been controversial for many years. Application of antiprogestin to nonovariectomized cycling guinea pigs leads to an inhibition of PGF2{alpha} secretion. This effect can be reversed by progestin treatment (5, 6). The conclusion drawn from these in vivo studies was that progesterone is the stimulator of uterine PGF2{alpha} secretion at the end of the cycle. However, in guinea pigs ovariectomized towards the end of the cycle 1) supplementation with progesterone alone leads to a minimal increase in PGF2{alpha} production, 2) injection of E2 alone leads to a small increase in PGF2{alpha} production, whereas 3) animals receiving progesterone and E2 in combination have greatly induced uterine PGF2{alpha} production (55). Our in vitro system supports the concept that E2 acting on a progesterone-primed uterus contributes towards the stimulus for increased uterine PGF2{alpha} production (7). This indicates that the observations in the intact animals are reconcilable with our in vitro study, as towards the end of the cycle E2 would also be present, and the two steroids would be acting in synergy.

In conclusion, our studies on purified epithelial and stromal cultures indicate a dual role for the stromal and epithelial compartments in the control of biologically active endometrial PGF2{alpha} (Fig. 8Go). The low levels of guinea pig uterine PGF2{alpha} seen during the early cycle in vivo can be explained by our in vitro data, whereby addition of MPA on an E2 primed system leads to a reduction in the synthesizing capacity of the epithelium, and an increase in the catabolic capacity of the stroma. This results in a net reduction of biologically active PGF2{alpha}. Conversely, the increasing levels of uterine PGF2{alpha} towards the end of the cycle can be explained by our observations that the addition of E2 to an MPA primed system leads to an increase in the synthesizing capacity of the epithelium.



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  		Figure 8. Proposed scheme for endometrial control of PGF2{alpha} levels in utero. In an E2 primed endometrium (left) the rise of progesterone leads to a decrease in PGF2{alpha} by down-regulating the synthesizing capacity (COX-2) of the epithelial compartment, and up-regulating the catabolic capacity (PGDH) of the stromal compartment. Conversely, in a progesterone-primed endometrium (right) the rise of E2 leads to an up-regulation in the synthesizing capacity (COX-2) of the epithelial compartment, which is not opposed by a corresponding increase in catabolic capacity (PGDH) of the stromal compartment.

 


   Acknowledgments
 
We thank Professors Dr. Freimut A. Leidenberger and Dr. Heinrich M. Schulte for their continual encouragement and support. We are also grateful to Dr. Matthias Schumacher for biotinylation of the ELISA tracers, Dr. Rodney Kelly for the kind gift of the PGF2{alpha} antibody, and Birthe Nitz for help with the cDNA library.


   Footnotes
 
1 This work is part of a doctoral study by K. E. Bracken, at the Department of Pre-Clinical Sciences, University of Leicester Medical School, University Road, Leicester, LE1 9HN, United Kingdom, and was supported by Deutsche Forschungsgemeinschaft Grant Bo 669/3-1. The nucleotide sequences of guinea pig COX-2 and PGDH cDNAs will appear in the EMBL, GenBank and DDBJ nucleotide sequence data bases under the accession numbers Y07896 and Y07953, respectively. Back

Received August 12, 1996.


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 Introduction
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 Results
 Discussion
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