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Regulation of 15-Hydroxyprostaglandin Dehydrogenase (PGDH) Gene Activity, Messenger Ribonucleic Acid Processing, and Protein Abundance in the Human Chorion in Late Gestation and Labor

Division of Obstetrics and Gynecology, John Hunter Hospital (T.Z.); and Discipline of Reproductive Medicine (R.F.J., T.Z.) and Mother’s and Babies Research Center (R.F.J., C.M.M., V.C., T.Z.), University of Newcastle, Newcastle, New South Wales 2310, Australia

Address all correspondence and requests for reprints to: Dr. Tamas Zakar, Division of Obstetrics and Gynecology, Mothers and Babies Research Center, John Hunter Hospital, Locked Bag 1, Hunter Region Mail Center, Newcastle, New South Wales 2310, Australia. E-mail: tzakar{at}mail.newcastle.edu.au.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
The prostaglandin (PG)-inactivating enzyme 15-hydroxyprostaglandin dehydrogenase (PGDH) is highly expressed in the chorion leave. To assess the involvement of PGDH in the regulation of intrauterine PG levels, we have determined the mechanisms that control chorionic PGDH expression in women at term and preterm labor. PGDH gene activity decreased at term and during normal labor. PGDH mRNA abundance also decreased at term due to changing splice variant distribution. Gene activity predicted PGDH mRNA abundance preterm and after normal labor, but not at term before labor. PGDH mRNA decayed rapidly in cultured tissues and was stabilized by transcriptional arrest. PGDH protein levels varied without being significantly different between the patient groups. PGDH mRNA levels predicted PGDH protein levels at term, but not preterm and after labor. PGDH gene activity, mRNA variant, and immunoreactive protein levels were not different between the preterm labor and preterm not in labor groups. Thus, PGDH mRNA is transiently down-regulated before term labor by a posttranscriptional mechanism(s). Protein turnover controls PGDH protein abundance at preterm and after normal labor. At term, PGDH protein levels become dependent on the rapidly turning over PGDH mRNA. This may allow rapid changes in PGDH protein abundance and uterotonic PG concentrations promoting labor.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
PROSTAGLANDINS (PGs) PRODUCED in the gestational tissues play central roles in human parturition by promoting cervical maturation, membrane rupture, and uterine contractions (1). The amnion membrane produces large amounts of PGE₂ (2), whereas the chorion laeve, situated between the amnion and the decidua, not only produces PGE₂ and PGF₂, but also controls the intrauterine concentration of active PGs by its high PG-metabolizing activity (3, 4, 5, 6, 7, 8, 9, 10). The intensive metabolism suggests that changing PG metabolic rates in the chorion may substantially alter bioactive PG levels in the uterus.

The first enzymatic step of PG metabolism is oxidation of the 15-hydroxyl group by the ß-nicotinamide adenine dinucleotide (NAD⁺)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) (11). This reaction inactivates PGs and constitutes the key step in PG metabolism. The nucleotide sequence of the PGDH mRNA and the amino acid sequence of the PGDH protein have been determined from human placenta (12, 13). The primary PGDH gene transcript undergoes alternative splicing that produces the 2-kb PGDH mRNA and two other splice variants with deletions of exon 6 and exons 5 and 6, respectively (14, 15). The splice variants are thought to correspond to a 3.4-kb RNA band on Northern blots and may be nonfunctional, because no immunoreactive PGDH protein of the corresponding size has been detected in cells expressing these variants.

In the fetal membranes, PGDH mRNA and protein have been localized to the chorion trophoblast layer. Cheung et al. (16) and Sangha et al. (17) reported no change in the localization of PGDH at term labor, and Germain et al. (4) and Van Meir et al. (18) saw no significant change in chorionic PGDH mRNA and activity levels with term labor. In contrast, others found a labor-associated increase in PGDH activity in chorion (9) and a decrease in PGDH mRNA levels in chorio-decidua (17) and cultured chorion cells (19). After preterm labor, PGDH mRNA and protein levels were lower compared with those at term (17, 18). In infection-associated preterm labor, no change (17) or a further decrease in enzyme activity and protein expression was reported (18). Changing regional distribution of PGDH activity and immunoreactive PGDH has also been detected in term chorion before and during labor (20).

Despite some disagreements between the results, the former studies suggest that chorionic PGDH expression is subject to complex and dynamic regulation during late pregnancy and labor. The nature, mechanism, and physiological significance of the regulatory changes are still unclear. In cell culture models, PGDH activity and mRNA abundance are influenced by progesterone, cortisol, cAMP, and intracellular calcium (21, 22, 23, 24). Transcription of the PGDH gene may be regulated by the transcription factors Ets, activating protein-1, and steroid receptors (25, 26). Furthermore, PGDH protein turnover is rapid and is modulated by agonists in cell culture (27). Thus, multiple mechanisms may alter PGDH expression in vitro, potentially explaining the variability of results when patient groups are compared or when individual differences within patient groups are analyzed.

The aim of the present study was to determine the contributions of gene activity, transcript processing, and enzyme protein levels to the chorionic expression of PGDH during late pregnancy and labor. We found evidence of transcriptional, posttranscriptional, and protein turnover-dependent regulation, which was strongly influenced by gestational age and labor. Our data suggest that rapid and transient changes at these steps of PGDH expression modulate bioactive PG levels and influence PG action in the late pregnant uterus.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Materials

Phenylmethylsulfonylfluoride, leupeptin, pepstatin A, aprotinin, sodium orthovanadate, dithiothreitol, 5,6-dichlorobenzimidazole riboside (DRB), glycerol, Cibacron Blue 3GA-agarose, NAD⁺, and Hanks’ balanced salts were obtained from Sigma-Aldrich Corp. (St. Louis, MO). Prestained low molecular weight standards (low range) for SDS-PAGE were obtained from Bio-Rad Laboratories (Hercules, CA). All other reagents were of analytical grade or the highest quality available.

Patients

Placentas with attached fetal membranes were obtained from 96 singleton pregnancies at John Hunter Hospital (Newcastle, Australia). Chorion leave was collected after elective cesarean section at term [term not in labor (TNL); 38.7 ± 1 wk of gestation; mean ± SD; n = 27] or preterm [preterm not in labor (PNL); 34.5 ± 1.6 wk; n = 20] and after spontaneous vaginal delivery at term [term labor (TL); 39.5 ± 1.1 wk; n = 26] or preterm [preterm labor (PTL); 32.9 ± 2.7 wk; n = 23]. The indications for preterm cesarean section included placenta previa, preeclampsia, insulin-dependent diabetes mellitus, and intrauterine growth retardation. Women who had received nonsteroidal antiinflammatory drugs within 3 d before delivery or with a history of infection, histological chorioamnionitis, severe asthma, or induced delivery were excluded from the study. Informed consent was obtained from all women enrolled in the study, and the Hunter Area Health Service and the University of Newcastle human ethics committees approved the use of these tissues.

Tissue collection

The reflected fetal membranes were isolated within 30 min of delivery. The amnion and chorio-decidua were separated by blunt dissection, and the chorion laeve was separated from the adherent decidua by sharp dissection, as previously described (28, 29). The uniform removal of decidual tissue by this procedure has been verified histologically in previous studies (29) and was monitored in the present study by examination of the chorion membrane preparations using an inverted phase contrast microscope (28). The chorion was snap-frozen in liquid nitrogen and stored at –80 C.

Isolation, purification, and RT of RNA

Total RNA from chorion tissue was prepared using TRIzol reagent (Invitrogen Life Technologies, Inc., Mt. Waverley, Australia) and was treated with deoxyribonuclease using Qiagen Mini-Spin columns and the Qiagen RNase-free DNase kit (Qiagen, Clifton Hill, Australia). RNA integrity was assessed by the visualization of 18S and 28S ribosomal RNA after agarose gel electrophoresis.

The Superscript First Strand Synthesis Kit for RT-PCR (Invitrogen Life Technologies, Inc.) was used to synthesize cDNA from 3 µg total RNA using random hexamers as primers.

Real-time PCR primer design

Primers (Table 1) were designed using the Primer Express version 1.0 computer program (Applied Biosystems, Foster City, CA). Primers measuring total PGDH mRNA were directed to an amplicon between open reading frame (ORF) positions 204–262, corresponding to a sequence spanning exons 1 and 2, common to all PGDH mRNA variants (15). Functional PGDH mRNA primers were directed to a sequence spanning exons 6 and 7, corresponding to an amplicon at ORF 651–707 of the full-length cDNA sequence (12). Primers for PGDH heterogeneous nuclear RNA (hnRNA) were directed to a sequence located between bases 2692–2760 within intron 1 of the PGDH gene (25). There are two identified and characterized splice variants of PGDH mRNA. We refer to these as variant 1, in which exon 6 has been removed (14), and variant two, in which both exon 5 and exon 6 have been removed (15). PGDH variant 1 mRNA primers, corresponding to an amplicon at ORF 490–543, were designed to span the junction of exons 5 and 7, whereas primers for PGDH mRNA variant 2 were designed to span the junction of exons 4 and 7, corresponding to an amplicon at ORF 409–466 of the respective sequences. Primers for ß-actin mRNA amplified a sequence located at ORF 878-1090 (30).

Real-time quantitative PCR, using an ABI PRISM 7700 sequence detector (Applied Biosystems, Branchburg, NJ) with SYBR Green detection, was used to determine the abundance of the target RNAs. Each 25-µl amplification mixture contained SYBR Green PCR Master Mix (Applied Biosystems), 400 nM forward primer, 400 nM reverse primer, and cDNA corresponding to 40 ng reverse transcribed total RNA. Threshold cycle numbers were determined after 40 amplification cycles, with triplicate amplifications performed for each of the target sequences. No-reverse transcriptase and no-template controls were included in the PCR analysis of each cDNA sample and primer pair to ensure that no fluorescent signal was generated by residual genomic DNA or primer interactions, respectively.

The conditions for constant relative amplification efficiency of the target vs. the reference mRNA sequences were established in preliminary experiments. The abundance of the reference sequence, ß-actin mRNA, did not differ between the patient groups. The homogeneity and predicted size of the amplification products were verified by melting curve analysis and gel electrophoresis, respectively, and the identity of the amplification products was confirmed by nucleotide sequencing.

Preparation of protein extracts

Frozen chorion (0.2 g) was homogenized in 2 ml ice-cold protein extraction buffer [PEB; 10 mmol/liter potassium phosphate (pH 7.4), 20% (vol/vol) glycerol, 1 mmol/liter EDTA, 1 mmol/liter dithiothreitol, 0.1 mmol/liter phenylmethylsulfonylfluoride, 1 µmol/liter leupeptin, 1 µmol/liter pepstatin A, 0.1 µmol/liter aprotinin, and 0.1 mmol/liter sodium orthovanadate] using a Polytron homogenizer (Brinkmann Instruments, Inc., Westbury, NY) on the maximal setting for two 30-sec bursts separated by a 30-sec cooling period. The homogenate was centrifuged at 10,000 x g for 10 min at 4 C, and the supernatant was removed to a fresh 1.5-ml microcentrifuge tube.

The protein concentration was determined in duplicate 50-µl aliquots of the 10-fold diluted supernatant using the bicinchoninic acid protein assay reagent kit (Pierce Chemical Co., Rockford, IL).

To increase signal intensity and decrease nonspecific immunoreactions on the Western blots, PGDH was extracted from the supernatants with Cibacron Blue 3GA-agarose (Sigma-Aldrich Corp.) (13) The Cibacron Blue-agarose (0.02 g) was rehydrated in 4 ml water overnight at 4 C. The rehydrated affinity matrix (0.1-ml packed volume) was then washed three times with 1 ml PEB and suspended in a volume of supernatant containing 2.5 mg protein. The suspension was rocked for 1 h at 4 C. The Cibacron Blue-agarose was then separated by centrifugation and washed twice with 0.3 ml PEB and once with 0.3 ml PEB containing 5 µmol/liter NAD⁺. Proteins were eluted twice by incubation in 50 µl 0.1 mol/liter Tris base and 0.5% (vol/vol) Triton X-100 for 15 min at 37 C. To the combined elutes (100 µl), 11 µl 10% (wt/vol) sodium dodecyl sulfate and 27.5 µl 5x loading dye [5% (wt/vol) sodium dodecyl sulfate, 312.5 mmol/liter Tris-HCl (pH 6.8), 50% (vol/vol) glycerol, 25% (vol/vol) 2-mercaptoethanol, and 0.0025% (wt/vol) bromophenol blue] were added. The samples were heated to 90 C for 10 min and stored at –80 C until electrophoresis.

Electrophoresis and immunoblotting of PGDH protein

Samples were separated in discontinuous SDS-polyacrylamide gel slabs with 12% separating gels (31) using a Mini-Protean II apparatus (Bio-Rad Laboratories). From each affinity-purified protein extract, 10- and 20-µl samples were loaded on the gel. The separated proteins were transferred to Hybond-C Extra nitrocellulose membranes (Amersham Biosciences, Uppsala, Sweden) overnight at 4 C following Towbin’s procedure (32). The nitrocellulose was blocked with 5% (wt/vol) skim milk in TBS-T [30 mmol/liter Tris-HCl (pH 7.4), 120 mmol/liter NaCl, and 0.1% (vol/vol) Tween 20] and was incubated overnight at 4 C with polyclonal rabbit PGDH antibody (Cayman Chemical Co., Ann Arbor, MI) diluted to 1:200 (2 µg/ml) in 5% skim milk in TBS-T. The blots were washed with TBS-T and incubated for 1 h at room temperature with horseradish peroxidase-labeled antirabbit IgG (Amersham Biosciences) diluted to 1:2000 with skim milk/TBS-T. Protein bands were visualized using the ECL Western Blotting Detection Kit (Amersham Biosciences). The blots were exposed to Kodak Biomax MS autoradiographic film (Eastman Kodak Co., Rochester, NY) at room temperature for 30 min. The films were developed and analyzed by densitometry using the Image computer program (ß Release 4.0.2, Scion, Frederick, MD) available online from the NIH web page. Protein loading was in the linear range of PGDH signal generation, as determined in pilot experiments. To compare PGDH protein levels between blots, calibrator samples from a pooled chorion preparation were loaded on each gel and used to normalize the results. Inter- and intraassay coefficients of variation in our Western blotting system were 9.85% and 9.93%, respectively.

Tissue incubation experiments

Tissue incubation experiments were conducted as described previously (33). RNA degradation rates and hnRNA processing rates for PGDH were determined by real-time PCR analysis after incubation at 37 C for 1, 2, 4, 6, and 24 h in the presence of the transcription inhibitor DRB at a final concentration of 80 µmol/liter. To determine the dynamics of PGDH hnRNA and mRNA abundance in vitro, tissue incubations were performed without DRB for 0, 2, 4, 6, 18, and 24 h. RNA extracted from the incubated tissues was analyzed as described above. The viability of chorion tissue during the incubation experiments was assessed by measuring lactate dehydrogenase release into the medium. Less than 5% of the total lactate dehydrogenase content of the tissues was released within 24 h in the presence or absence of DRB, indicating the high viability of chorion under the conditions employed.

Data analysis

The relative abundance of mRNAs was calculated using the formula: relative abundance = 2^–Ct, where Ct is the difference between the threshold cycles (Ct) of the test sequence and the reference sequence (ß-actin mRNA). Computer software provided with the ABI PRISM 7700 sequence detector was used for this purpose. Relationships were analyzed by Robust Regression (STATA version 8.0, Stata Corp., College Station, TX) of logarithmically transformed data. Parametric group comparisons were performed on logarithmically transformed data using one-way ANOVA, followed by Tukey’s multiple comparisons test. Where appropriate, nonparametric group comparisons were performed using the Kruskal-Wallis ANOVA, followed by Dunn’s multiple comparisons test. The results of the tissue incubation studies were analyzed by two-way ANOVA (mixed model, with repeated measures), followed by Tukey’s test for multiple comparisons if significant F values were obtained. Power tests were conducted using nQuery Advisor Release 5.0 (Statistical Solutions, Saugus, MA). In all statistical analyses, P < 0.05 was considered significant.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
PGDH mRNA splice variants in the chorion at late gestation and labor

The levels of total, functional, and nonfunctional splice variants of PGDH mRNA were determined, relative to ß-actin mRNA, in the PNL, TNL, and TL groups (Table 2). Total PGDH mRNA abundance did not differ significantly among the groups, indicating that the combined abundance of PGDH mRNA splice variants did not change with advancing gestation and labor. The relative abundance of the two nonfunctional splice variants was very low, and the median values were not different between the patient groups. The abundance of the functional PGDH mRNA, however, was significantly (P < 0.05) lower in the TNL group than in the PNL and TL groups (P < 0.05; Table 2). Thus, functional PGDH mRNA splice variant levels decreased transiently and selectively before TL.

hnRNA is the precursor of mature mRNA, and hnRNA levels serve as a measure of gene activity in fresh tissues (33, 34) and cultured cells (35, 36). We determined PGDH hnRNA levels in the PNL, TNL, and TL groups using quantitative real-time RT-PCR with intron-specific primers (Table 1). PGDH hnRNA levels were lower (P < 0.05) in the TNL group than in the PNL group, and there was no significant difference between the TNL and TL groups (Table 2). Thus, the PGDH gene transcription rate decreases in the chorion at term before labor, and labor has no significant further influence on PGDH gene activity (Table 2).

The relationships between PGDH hnRNA and mRNA levels in individual tissues were examined by regression analysis in the PNL, TNL, and TL groups. In PNL patients, PGDH hnRNA was a significant predictor of both the total (Fig. 1A; P < 0.001) and the functional (Fig. 1B; P < 0.001) PGDH mRNAs, and total PGDH mRNA levels predicted the levels of functional PGDH mRNA (Fig. 1C; P < 0.001). In preterm chorion, therefore, PGDH gene activity significantly determined the levels of both total and the functional PGDH mRNAs, and a consistent proportion of the total PGDH mRNA was processed into the functional splice variant. In the TNL group, PGDH hnRNA levels significantly predicted total PGDH mRNA levels (P < 0.001; Fig. 2A), but did not predict the levels of the functional PGDH mRNA (Fig. 2B). Moreover, no significant regression was detected between total and functional PGDH mRNA abundance in this group (Fig. 2C). These relationships suggested that before term labor, functional PGDH mRNA abundance was not determined by PGDH transcriptional activity, but was controlled at a posttranscriptional step(s). In the TL group, PGDH hnRNA abundance predicted the level of both the total (P < 0.005; Fig. 3A) and the functional (P < 0.001; Fig. 3B) PGDH mRNA, and total PGDH mRNA abundance predicted the level of the functional mRNA (P < 0.001, Fig. 3C). Thus, after term labor, the relationships between PGDH hnRNA and PGDH mRNAs were restored to those seen in the PNL group.

View larger version (15K): [in this window] [in a new window]   FIG. 1. Relationships among the levels of PGDH hnRNA, total PGDH mRNA, and functional PGDH mRNA in chorion membranes before term in the PNL group. The relative abundance of PGDH hnRNA and that of total and functional PGDH mRNAs were measured by quantitative real-time RT-PCR. Relationships between PGDH hnRNA and total PGDH mRNA levels (A), between PGDH hnRNA and functional PGDH mRNA levels (B), and between total and functional PGDH mRNA levels (C) were determined by Robust Regression with log-transformed data. Each point represents an individual patient.

View larger version (17K): [in this window] [in a new window]   FIG. 2. Relationships among the levels of PGDH hnRNA, total PGDH mRNA, and functional PGDH mRNA in chorion membranes at term in the TNL group. A, Relationship between PGDH hnRNA and total PGDH mRNA levels; B, relationship between PGDH hnRNA and functional PGDH mRNA levels; C, relationship between total and functional PGDH mRNA levels. Other conditions are described in Fig. 1.

View larger version (17K): [in this window] [in a new window]   FIG. 3. Relationships among PGDH hnRNA, total PGDH mRNA, and functional PGDH mRNA levels in chorion membranes in the TL group. A, Relationship between PGDH hnRNA and total PGDH mRNA levels; B, relationship between PGDH hnRNA and functional PGDH mRNA levels; C, relationship between total and functional PGDH mRNA levels. Other conditions are described in Fig. 1.

The processing of hnRNA and the degradation of mRNA are critical steps in mRNA turnover. We investigated the processing of PGDH hnRNA and the degradation of PGDH mRNA by incubating chorion tissue in the presence of DRB, a transcriptional inhibitor. Real-time quantitative RT-PCR was employed to monitor the abundance of PGDH hnRNA and (functional) PGDH mRNA at the different time points. Chorion membranes collected from women after elective cesarean delivery displayed a slow decrease in functional PGDH mRNA relative abundance (estimated t_1/2, 24 h; Fig. 4A; n = 3). After labor at term, functional PGDH mRNA showed a tendency for an increased degradation rate (t_1/2, 14.5 h; Fig. 4B; n = 3); however, the difference between the half-lives did not reach statistical significance. In both the TNL and TL groups, PGDH hnRNA relative abundance decreased rapidly by up to 85–90% in the first 2 h of incubation and remained low. Thus, PGDH hnRNA processing rates at term before and after labor are fast, with no change associated with labor.

View larger version (14K): [in this window] [in a new window]   FIG. 4. A and B, Kinetics of PGDH mRNA decay and hnRNA processing in tissue incubations. Chorion membranes delivered by term elective cesarean section (A) and TL (B) were incubated in the presence of the transcriptional inhibitor, DRB. The relative abundance of PGDH hnRNA and that of functional PGDH mRNA were determined by quantitative real-time RT-PCR. Data are expressed as a percentage relative to the zero hour value. C, Dynamics of PGDH hnRNA and mRNA abundance in tissue incubations. Chorion membranes delivered by elective term cesarean section were incubated without transcriptional inhibitor. In all panels, the mean ± SE of three independent experiments is shown. Fifty percent values are indicated by dashed lines, and asterisks indicate significant differences (P < 0.025) vs. the zero hour points.

PGDH protein expression in late gestation and term labor

The level of PGDH protein was measured in the PNL, TNL, and TL groups by immunoblotting. In all chorion samples, an immunoreactive band with the size of PGDH (molecular mass, 29,000) (12, 13) was observed (Fig. 5A). The specificity of the immunodetection was confirmed using purified rabbit IgG instead of the primary antibody (Fig. 5B) and by preadsorption of the primary antibody with the immunizing peptide (Fig. 5C). Immunoreactive PGDH protein levels were variable in individuals, but the mean values did not differ significantly among the patient groups, suggesting that overall PGDH protein abundance did not change with advancing gestation and labor (Table 3).

View larger version (62K): [in this window] [in a new window]   FIG. 5. PGDH protein detection by immunoblot. Lane 1, 28.8-kDa molecular mass marker; lane 2, pooled chorion tissue extract; lane 3, extract of an individual chorion delivered by TL; lanes 4 and 5, extracts of individual chorion membranes delivered by TL and PNL, respectively. A, Immunodetection using PGDH primary antibody; B, immunodetection using rabbit IgG; C, immunodetection with PGDH primary antibody in the presence of excess immunizing peptide. The same amounts of extracted protein from the same chorion preparations were processed in corresponding lanes of A, B, and C.

We have explored the relationships between functional PGDH mRNA and protein expression by regression analysis in which mRNA levels were considered the independent variable, and protein levels were considered the dependent variable. In the PNL and TL groups, PGDH mRNA levels did not predict the levels of PGDH protein (Fig. 6, A and C), but in the TNL patients, functional PGDH mRNA was a significant (P < 0.01) predictor of PGDH protein levels (Fig. 6B). Thus, the PGDH mRNA concentration significantly determined the abundance of PGDH enzyme protein before labor at term, but not preterm or after term labor.

View larger version (15K): [in this window] [in a new window]   FIG. 6. Relationships between functional PGDH mRNA and immunoreactive PGDH protein levels. Chorion tissues were delivered by preterm and term elective cesarean section (PNL in A and TNL in B, respectively) and after labor at term (TL in C). Relationships were determined by Robust Regression.

We also examined the effect of (idiopathic) PTL on the levels of PGDH mRNA and protein. For this analysis, chorion tissue was collected from 24 women who delivered (32.9 ± 0.55 wk; mean ± SD) in the absence of intrauterine infection or inflammation. The PTL group showed no significant differences in the relative abundance of total (mean ± SD, 0.846 ± 0.132), functional (0.826 ± 0.169), or nonfunctional splice variants of PGDH mRNA (0.009 ± 0.002 and 0.002 ± 0.000, respectively) compared with the corresponding values in the PNL group (listed in Table 2; by unpaired t test with a Welch correction). Similarly, neither PGDH hnRNA nor PGDH protein levels were significantly different between the PTL and PNL groups (0.200 ± 0.005 and 0.816 ± 0.164, respectively, for PTL; values for PNL are listed in Table 2). We characterized the relationships between PGDH hnRNA and PGDH mRNA and between PGDH mRNA and PGDH protein levels in the PTL patients. As illustrated in Fig. 7, A and B, respectively, PGDH hnRNA levels significantly predicted both total (P < 0.001) and functional (P < 0.001) PGDH mRNA abundance. Furthermore, total PGDH mRNA levels significantly (P < 0.001) predicted the levels of the functional PGDH mRNA (Fig. 7C). However, there was no significant regression between functional PGDH mRNA and PGDH protein abundance (Fig. 8). These relationships indicated that in the chorion after PTL, PGDH mRNA abundance was controlled by the transcriptional rate, and PGDH protein levels were not dependent on PGDH mRNA abundance.

View larger version (16K): [in this window] [in a new window]   FIG. 7. Relationships among PGDH hnRNA, total PGDH mRNA, and functional PGDH mRNA levels in the PTL group. A, Relationship between PGDH hnRNA and total mRNA; B, relationship between PGDH hnRNA and functional mRNA; C, relationship between total and functional PGDH mRNA. Relationships were tested by Robust Regression. Other details are described in Fig. 1.

View larger version (17K): [in this window] [in a new window]   FIG. 8. The relationship between functional PGDH mRNA and PGDH protein levels in the PTL group. The relationship was tested by Robust Regression.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

We included in this study a patient group who delivered by cesarean section before term in the absence of labor (PNL group). Importantly, the various indications of the elective preterm cesarean sections had no apparent influence on PGDH mRNA levels (data not shown). By comparing this group with the TNL group, we assessed the temporal change in PGDH mRNA expression in late pregnancy and found decreasing functional, but not total, PGDH mRNA abundance with advancing gestation. Taken together, the analysis of PGDH mRNA splice variant expression suggested that a transient decrease in functional PGDH mRNA abundance occurred at term before labor, which was caused predominantly by changing splice variant distribution.

Evaluation of PGDH hnRNA levels revealed a significant decrease in the gene transcription rate at term before labor (TNL vs. PNL). The decrease, however, did not cause a significant decrease in total PGDH mRNA abundance. The relationships among hnRNA, total mRNA, and functional mRNA abundance in individual tissues indicated that a transcription-driven mechanism(s) controlled functional PGDH mRNA levels in the PNL and TL groups. In these patients, hnRNA levels predicted total and functional mRNA levels, and total PGDH mRNA levels predicted functional PGDH mRNA levels. In the TNL group, however, the levels of hnRNA predicted total, but not functional, PGDH mRNA levels, and there was no relationship between total and functional PGDH mRNA abundance. This suggested that a posttranscriptional mechanism(s) controlled the functional PGDH mRNA concentration in the chorion before TL.

PGDH protein levels were not different in the PNL, TNL, and TL groups, in agreement with previous findings by Sangha et al. (17). We also confirmed the substantial variability in immunoreactive PGDH protein levels between individuals in all patient groups. Assuming a requirement of 80% power, the variability limited the detection of differences between the means to an ANOVA effect size of 0.169 or higher, using logarithmically transformed data. The effect size in our protein dataset was 0.045 (n = 20/group), indicating that smaller differences between mean protein abundance values might have remained undetected. A several-fold increase in patient numbers, as indicated by the power tests, was unfeasible.

There was no significant relationship between PGDH protein and functional mRNA levels in individual tissues of the PNL and TL groups. This suggests that before term and after term labor, PGDH abundance is controlled predominantly by protein turnover. In the TNL group, however, PGDH mRNA levels significantly predicted the abundance of the cognate protein, implying that PGDH mRNA abundance may control PGDH enzyme protein levels before labor onset at term.

The kinetics of PGDH mRNA turnover in term chorion indicated a tendency for increased degradation after labor. This agrees with the observation of Pomini et al. (19), who found higher PGDH mRNA levels before than after labor in chorion explants cultured for 24–48 h. Furthermore, the spontaneous decrease in functional PGDH mRNA abundance during incubation and the stabilization of this mRNA in transcriptionally arrested tissues suggest that PGDH mRNA turnover at term is rapid and is facilitated by factors dependent on continuous transcription. The fast turnover of PGDH mRNA may lead to fast changes in PGDH protein expression, particularly in the TNL group, as suggested by the relationship shown in Fig. 6B. Therefore, it is reasonable to surmise that the posttranscriptional processing and/or degradation rate of PGDH gene products influence PGDH expression, PG metabolism, and bioactive PG concentrations in the uterus, specifically at term labor onset.

By comparing the PNL and PTL groups, we were able to assess the effect of (idiopathic) PTL on chorionic PGDH expression. These comparisons showed no difference between PGDH gene activity, total mRNA abundance, splice variant levels, and protein abundance in the two groups. The analysis of relationships in individuals indicated transcriptionally controlled functional PGDH mRNA levels and protein turnover-dependent PGDH protein levels in both groups. Thus, idiopathic PTL is not associated with changing chorionic PGDH mRNA and protein expression. Such a conclusion, however, must be made with caution, because PGDH mRNA regulation may change transiently before PTL, similarly to what occurs before labor at term. The changes in PGDH expression before PTL are not practical to investigate, because it is not possible to obtain chorion membranes from women with impending PTL.

Overall, our data demonstrate that chorionic PGDH mRNA expression switches from transcriptional to posttranscriptional control before TL. In concert with this change, PGDH protein expression becomes dependent on PGDH mRNA abundance. Because the turnover of chorionic PGDH mRNA is fast at term, transient changes in PGDH mRNA processing and/or degradation may rapidly alter enzyme levels. It is well documented that the PG synthetic capacity of the fetal membranes is high and increases further in late gestation (1, 33, 34). The tight and dynamic regulation of PGDH abundance and PG metabolism is therefore important for birth to occur in an orderly physiological manner.

	Acknowledgments

 
We gratefully acknowledge the help provided by Dr. Andrew Bisits during the statistical evaluation of data.

	Footnotes

 
This work was supported by a Hunter Medical Research Institute research award (to University of Newcastle and the Mothers and Babies Research Center) and New South Wales Health through the Hunter Medical Research Institute. A scholarship for R.F.J. was provided by Research Higher Degrees, University of Newcastle, and contributed by Profs. Roger Smith and David Smith.

Abbreviations: DRB, 5,6-Dichlorobenzimidazole riboside; hnRNA, heterogeneous nuclear RNA; NAD⁺, ß-nicotinamide adenine dinucleotide; ORF, open reading frame; PEB, protein extraction buffer; PG, prostaglandin; PGDH, 15-hydroxyprostaglandin dehydrogenase; PNL, preterm not in labor; PTL, preterm labor; TBS-T, 30 mmol/liter Tris-HCl (pH 7.4), 120 mmol/liter NaCl, and 0.1% (vol/vol) Tween 20; TL, term labor; TNL, term not in labor.

Received March 30, 2004.

Accepted July 27, 2004.

	References

Top Abstract Introduction Patients and Methods Results Discussion References

 

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