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The Journal of Clinical Endocrinology & Metabolism 87(11):5274 –5282
Copyright © 2002 by The Endocrine Society
doi: 10.1210/jc.2002-020521
Expression of Prostaglandin I2 Synthase, but Not
Prostaglandin E Synthase, Changes in Myometrium of
Women at Term Pregnancy
D. GIANNOULIAS, N. ALFAIDY, A. C. HOLLOWAY, W. GIBB, M. SUN, S. J. LYE,
J. R. G. CHALLIS
AND
Canadian Institutes of Health Research in Human Development, Child and Youth Health, Departments of Physiology and
Obstetrics and Gynecology, University of Toronto (D.G., N.A., A.C.H., S.J.L., J.R.G.C.), Toronto, Canada M5S 1A8;
Department of Obstetrics and Gynecology, Ottawa Hospital (W.G., M.S.), Ottawa, Canada K1H 8L6; and Samuel Lunenfeld
Research Institute, Mount Sinai Hospital (S.J.L.), Toronto, Ontario, Canada M5G 1X5
Prostaglandins (PGs) act as potent uterotonins at the time of
labor. Prostaglandin E synthase (PGES) is responsible for the
formation of PGE2, a uterotonin. PGI2 is synthesized by the
prostaglandin I synthase enzyme (PGIS) and contributes to
relaxation in the lower uterine segment. We examined the
expression of membrane-bound PGES and PGIS in myometrium from pregnant women during preterm and term labor.
Tissues were collected from the lower uterine segment from
preterm no labor, preterm labor, term no labor, and term labor
patients and used for immunohistochemistry and Western
blot analysis using specific antibodies. Immunoreactive (ir-)
PGES and PGIS proteins were localized to the cytoplasm of
myocytes of the myometrium and vascular smooth muscle
cells. Ir-PGES was also detected in vascular endothelial cells.
P
ROSTAGLANDINS (PG) play many key roles in mediating physiological processes during pregnancy and
labor, including myometrial relaxation and contractility, regulation of utero-placental blood flow, and cervical ripening
(1–3). Therefore, examination of PG synthesis within the
pregnant uterus is important to elucidate PG effects locally
within myometrium from pregnant women. Prostaglandin
E2 (PGE2) is a predominant PG in intrauterine tissues (1–3)
and has been shown to play an important role in promoting
uterine contractility at the time of labor (4, 5). The synthesis
of PGE2 is dependent not only on the actions of PGHS (types
I and II), but also on the expression and activity of specific
PGE synthases (PGES) (6, 7). Similarly, the synthesis of PGI2
depends upon the action of a specific prostaglandin I synthase enzyme (PGIS), which converts PGH2 to PGI2 (8). PGI2
is the most abundantly produced PG in the myometrium
(8 –10) and during pregnancy is important in reducing myometrial contractility and regulating uterine and placental
blood flow (11, 12). Therefore, changes in the expression or
activity of PGES and PGIS enzymes during pregnancy and
labor could lead to altered synthesis of the primary PGs,
PGE2 and PGI2.
PGES is a member of a protein superfamily consisting of
membrane-associated proteins that are involved in eicoAbbreviations: cPGES, Cytosolic prostaglandin E synthase; DTT, dithiothreitol; ir-, immunoreactive; mPGES, membrane-bound prostaglandin E synthase; PG, prostaglandin; PGES, prostaglandin E synthase;
PGIS, prostaglandin I synthase enzyme.
Western blot analyses revealed a predominant protein band of
180 kDa, and a second 16-kDa band for ir-PGES and 56-kDa
band for ir-PGIS. There was no significant change in ir-PGES
protein (180 or 16 kDa) or mRNA levels with preterm or term
labor or gestational age. There was a significant decrease in
PGIS mRNA and protein with advancing gestational age. We
conclude that the gestational age decrease in the inhibitory
PGIS is consistent with lessening of its influence in myometrium at the time of labor. The lack of change in PGES indicates that alterations at other points along the pathway of
arachidonic acid metabolism may be of greater importance in
affecting local changes in PGE2. (J Clin Endocrinol Metab 87:
5274 –5282, 2002)
sanoid and glutathione metabolism (the MAPEG family)
(13). There are two distinct forms of PGES that have been
identified requiring glutathione as a cofactor: cytosolic PGES
(cPGES) and membrane-bound PGES (mPGES). Cytosolic
PGES is identical to p23, which is reportedly the weakly
bound component of the steroid hormone receptor/hsp90
complex and is constitutively expressed, unaltered by proinflammatory stimuli, in various cells and tissues (14). Jakobsson et al. (6) identified and characterized the human microsomal or membrane-bound PGES enzyme and revealed that
mPGES is inducible by IL-1␤ in lung carcinoma A549 cells.
In addition, mPGES colocalized with both PGHS isozymes in
the perinuclear envelope; however, mPGES was functionally
coupled with PGHS-II in marked preference to PGHS-I (15).
In sheep placenta, levels of PGES increase progressively
through pregnancy (15a). Recent studies have localized
mPGES to human fetal membranes and specified strong
staining in the epithelial layers of the amnion and chorionic
trophoblast layer (16, 17). There was no difference in mPGES
protein between the preterm or term groups with or without
labor for either amnion or chorion (17). There is no information, however, concerning levels of PGES in myometrium
at the time of labor.
PGI synthase is a membrane-bound hemoprotein that has
been localized by immunohistochemical techniques to endothelial cells and both vascular and nonvascular smooth
muscle cells (8, 18). PGI2 inhibits human and sheep myometrial activity in vivo and in vitro (19, 20). Interestingly, PGIS
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Giannoulias et al. • PGES and PGIS in Myometrium
is expressed in human myometrial smooth muscle (21), particularly the myofibrils (22), and seems likely to change during pregnancy as myometrial production of PGI2 rises (19).
However, it is not known whether the levels of PGIS in
myometrium from pregnant women change in association
with labor. We hypothesized that labor at term and preterm
would be associated with decreased expression of PGIS,
thereby lessening the inhibitory influence of PGI2 on myometrium, and with increased expression of PGES, to increase
production of the potentially stimulatory PGE2. Therefore,
we examined the specific localization pattern of PGES and
PGIS in myometrium from pregnant women and determined
whether the expression of these enzymes changed with labor
at preterm and term.
Materials and Methods
Tissue collection
Patient consent and ethical approval were obtained before the onset
of the study and tissue collection, according to the guidelines of Mount
Sinai Hospital (Toronto, Canada) and University of Toronto. Tissues
were collected from the upper edge of the lower uterine segment during
cesarean section deliveries and either snap-frozen in liquid nitrogen for
protein extraction or slow-frozen for use in immunohistochemistry or in
situ hybridization. Myometrial samples were then separated into four
groups: preterm (28 –36 wk), not in labor (n ⫽ 8) and in labor (n ⫽ 4);
term (38 – 40 wk) not in labor (n ⫽ 8), and labor (n ⫽ 6). Placenta was
collected at term (37– 42 wk) either during spontaneous labor (n ⫽ 5) or
at elective cesarean section (no labor; n ⫽ 4; fetal distress). Labor was
defined as the presence of regular uterine contractions every 1–2 min and
lasting for 1 min, resulting in cervical dilatation of 8 –10 cm. None of the
patients in this study had received PG or oxytocin. Cesarean sections
were either scheduled (nonlaboring patients) or performed because of
fetal or maternal complications (laboring patients). Indications for cesarean section were preterm no labor (fetal anomalies, n ⫽ 3; maternal
condition, n ⫽ 3; triplets, n ⫽ 2), preterm labor (fetal anomalies, n ⫽ 1;
breech, n ⫽ 3), term no labor (previous cesarean section, n ⫽ 4; breech,
n ⫽ 3), and term labor (fetal distress, n ⫽ 4; failure to progress, n ⫽ 2).
Immunohistochemistry
Immunoreactive PGES was localized in frozen samples of human
myometrium using a rabbit antihuman polyclonal antibody raised
against a peptide corresponding to amino acids 59 –75 (CRSDPDVERSLRAHRND) of human mPGES (Cayman Chemical, Ann Arbor, MI) at
a dilution of 1:50 in antibody dilution buffer. Immunoreactive (ir-) PGIS
was localized in myometrial tissue using a mouse antihuman monoclonal antibody raised against human PGIS (Cayman Chemical) at a
dilution of 1:50 in antibody dilution buffer. A monoclonal mouse antismooth muscle ␣-actin antibody (A2547, Sigma, St. Louis, MO) diluted
1:3000 in antibody dilution buffer was raised against the NH2-terminal
synthetic decapeptide of ␣-smooth muscle actin and was used to identify
smooth muscle cells. Immunohistochemistry was conducted as described previously (23). Negative control sections were treated with
PGES and PGIS antibodies that had been preabsorbed with the appropriate antigen overnight at 4 C before application to the tissue sections
(23).
Dual immunofluorescence
Tissue sections were rehydrated in serial dilutions of alcohol (100%,
90%, 70%, and 50%) and washed in PBS. Nonspecific binding of antibodies was blocked with 1% BSA in PBS for 2 h at room temperature.
The samples were then incubated with rabbit antihuman mPGES (1:50),
mouse anti-smooth muscle ␣-actin (1:3000), and/or mouse antihuman
PGIS (1:50) and placed in a 1% BSA solution containing 0.3% Triton
X-100. Tissue sections were incubated overnight (18 –24 h) with the
primary antibodies at 4 C. After the incubation period, the sections were
washed three times in 0.1 m PBS. The secondary antibodies were added
and incubated at room temperature for 2 h. The secondary antibodies
J Clin Endocrinol Metab, November 2002, 87(11):5274 –5282 5275
used were fluorophore- and biotin-labeled Alexa Fluor 568 goat antirabbit IgG (red) and Alexa Fluor 488 goat antimouse IgG (green) (Molecular Probes, Inc., Eugene, OR) at a 1:200 dilution in a 1% BSA solution.
Samples were washed again in PBS and then dehydrated in serial dilutions of alcohol (50%, 70%, 90%, and 100%). Anti-fading reagent [pphenylendiamine (1 mg/ml), 50% glycerol, and 50% PBS) was added to
the tissue sections, and coverslips were applied before analysis.
Microscopic analysis
Tissue sections were analyzed under a fluorescent Optiphot-2 microscope (Nikon, Melville, NY) using a green filter to visualize Alexa
Fluor 488 and a red filter to visualize Alexa Fluor 568. A Sensicam 12-bit
cooled monochrome imaging camera (Cooke, Inc., Auburn Hills, MI)
was used to take a digital photograph of the section using Sensicontrol
4.02 software (Cooke, Inc.), and this was visualized on a computer and
then exported into Coreldraw Corel (Eastman Kodak Co., Rochester,
NY) to produce generated color images as described previously (23).
These images were superimposed to obtain the localization patterns of
PGES with ␣-actin and PGIS.
Protein extraction and Western blot analysis
Protein was extracted from frozen myometrial and placental samples
as previously described (23). Homogenates were transferred to Eppendorf tubes (1–5 ml) and centrifuged at 15,000 ⫻ g at 4 C for 15 min to
remove tissue debris. Supernatants were collected and transferred to
fresh tubes, and protein concentrations were determined using the Bradford protein assay (24) with BSA diluted in protein assay dye reagent as
standard (Bio-Rad Laboratories, Inc., Hercules, CA). Concentrations of
protein samples were quantified by linear regression analysis from the
standard curve.
Myometrial protein (50 ␮g) was solubilized, resolved onto a 10%
(PGIS) or 12% (PGES) bis-acrylamide gels/4% stacking gels, and transferred onto a nitrocellulose membrane as described previously (23). The
PGES and PGIS antibodies (both from Cayman Chemical) were diluted
1:200 and 1:500, respectively, in blocking solution. The blots were then
incubated with either antirabbit IgG or antimouse IgG secondary antisera conjugated to horseradish peroxidase (1:2000; Amersham Pharmacia Biotech, Baie d’Urfe, Canada) in blocking solution. The relative
intensities of immunoreactive protein signals were quantified using
computerized image analysis (MCID Imaging Research, St. Catharines,
Canada). Protein bands were digitized, and the mean pixel density for
each band was analyzed to obtain relative OD units for each protein. The
blots were stripped (0.1 m glycine, pH 2.7; Sigma) and reprobed with the
G␤ antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) as an
internal control for protein loading. The G␤ antibody detects a 40-kDa
protein that does not change in association with gestational age or labor
at term or preterm (25). Because initial studies revealed that the major
PGES band in myometrium was 180 kDa, we compared myometrium
with placenta, a tissue with high PGES activity (6) previously shown to
express predominantly the 16-kDa isoform, using the same extraction
techniques.
In situ hybridization for PGES and PGIS
Samples of human myometrium were collected and stored in OCT
embedding medium (Sakahura Fine Chemical Co., Torrance, CA). These
specimens were cut into sections (12 ␮m) on a cryostat and postfixed in
4% paraformaldehyde for 5 min, followed by two washes in PBS, and
then dehydrated by immersion for 2 min in 70% ethanol. Solutions for
tissue preparation and in situ hybridization were prepared with 0.01 m
PBS or ddH2O that had been treated previously with diethyl pyrocarbonate (Sigma) to abolish ribonuclease activity.
The 50-mer antisense probe (CTT CTT CCG CAG CCT CAC TTG
GCC CGT GAT GAT GGC CAC CAC GTA CAT CT) used for in situ
hybridization of PGES was complementary to bases 95–144 of the human
PGES gene (26). The PGIS probe (GTG CCA CTG TAG AAA TGA TAT
GAC CAA CCC CCG TCC ATA CAG TGG) was complementary to
bases 181–226 of the human PGIS gene (GenBank accession no. 030508).
The probes were synthesized in the molecular biology facility at University of Ottawa using an Oligo 1000 DNA synthesizer (Beckman,
Fullerton, CA) and purified by cartridge purification. Hybridization
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Giannoulias et al. • PGES and PGIS in Myometrium
FIG. 1. Expression of ␣-actin in myometrium from women in late pregnancy. A, Comparison of ␣-actin in myometrium from all four groups of
patients determined by Western blotting: PT/NL, preterm no labor (n ⫽ 8);
PT/L, preterm labor (n ⫽ 4); T/NL, term
no labor (n ⫽ 8); and T/L, term labor
(n ⫽ 6). B, Immunohistochemical localization of ir-␣-actin and a representative hematoxylin and eosin (H&E) stain
of the myometrium from a term nonlaboring patient (T/NL) and a previous
cesarean section patient (PCS). C, Representative Western blot and comparison of ␣-actin in myometrium from term
nonlaboring (T/NL) patients and from
patients who underwent a previous cesarean section (PCS). The histograms
represent the relative OD units of ␣actin protein. Data were analyzed by
two-way ANOVA (␣ ⫽ 0.05). There were
no significant differences in ␣-actin content between the groups shown. All values are the mean ⫾ SEM.
with corresponding sense probes (prepared in a similar fashion) served
as the negative control.
Probes were labeled using terminal deoxynucleotide transferase (Life
Technologies, Inc., Burlington, Canada) and [␣-35S]deoxy-ATP (12.5
mCi/ml; NEN Life Science Products-DuPont Canada, Inc., Mississauga,
Canada). Labeled probe was purified using a SORB 20 column (NEN Life
Science Products-DuPont Canada, Inc.) and was used at a concentration
of 5000 cpm/␮l. Slides were removed from the ethanol and allowed to
air-dry at room temperature in a fumehood. The slides were incubated
overnight at 45 C in a moist chamber with the radiolabeled oligonucleotide probe in hybridization buffer. Hybridization buffer was composed of 4⫻ SSC [1⫻ SSC ⫽ 150 mm sodium chloride and 15 mm sodium
citrate (Sigma)], 50% deionized formamide (Life Technologies, Inc.), 10%
dextran sulfate (Amersham Pharmacia Biotech), 25 mm sodium phosphate (pH 7.0), 1 mm sodium pyrophosphate, 200 ␮g acid-alkali hydrolyzed salmon sperm DNA/ml, 100 ␮g polyadenylic acid/ml, 120 ␮g
heparin/ml, 40 mm dithiothreitol (DTT; Sigma), and 5⫻ Denhardt’s
solution.
Negative control slides were incubated with sense probes. After the
overnight incubation, slides were washed in 1⫻ SSC (containing 10 mm
DTT) at room temperature for 20 min, then in 1⫻ SSC (with DTT) at 55
C for 45 min and rinsed in 1⫻ SSC, 0.1⫻ SSC, and ddH2O at room
temperature for 10 sec each. Slides were dehydrated in ethanol (70% and
95% for 10 sec each), air-dried for 2 h, and exposed to x-ray film (Biomax,
Eastman Kodak Co.) using standard methods. Linearity was established
by simultaneous exposure of the film to a 14C-labeled standard (RPA504,
Amersham Pharmacia Biotech). The autoradiograms were analyzed using computerized analysis software (Image Research, St. Catherines,
Canada). Relative OD values were obtained for slides of three or four
sections per animal.
Statistical analysis
Results from Western blotting analysis and in situ hybridization are
presented as the mean ⫾ sem for each patient group; data were analyzed
using a two-way ANOVA. Statistical significance was set at P ⱕ 0.05. A
Giannoulias et al. • PGES and PGIS in Myometrium
J Clin Endocrinol Metab, November 2002, 87(11):5274 –5282 5277
FIG. 2. Ir-PGES and ir-PGIS staining in human myometrium. A, Ir-PGES at term (no labor) cesarean section. B, Ir-PGES at term (no labor)
in association with a myometrial blood vessel, viewed at higher magnification. C, Section stained with preabsorbed primary antibody for PGES.
D, Ir-PGIS at term (no labor). E, Ir-PGIS at term (no labor) at higher magnification. F, Section stained with preabsorbed primary antibody for
PGIS. Brown color indicates positive staining. Photographs were taken at a magnification of ⫻200 and ⫻400. Scale bar, 25 ␮m (⫻200) and 12.5
␮m (⫻400). SM, Smooth muscle; EN, endothelial cells; VSM, vascular smooth muscle.
t test was used if the two-way ANOVA indicated a significant main
effect. Calculations were performed using SigmaStat (Jandel Scientific
Software, San Rafael, CA).
Results
Expression of ␣-actin in myometrium from pregnant women
To compare the proportions of myocytes in different tissue
specimens, we measured the content and distribution of
␣-actin in samples of myometrium from different patient
groups. There were no significant differences observed in the
␣-actin contents of myometrium from women in all four
groups (Fig. 1A). Furthermore, there was no obvious change
in the immunohistochemical localization of ␣-actin or in the
proportion of ␣-actin-positive cells in myometrium from
women at term in the absence of labor with or without
previous cesarean section (Fig. 1B). There was no significant
difference in the content of ␣-actin in lower segment myometrium at term in the absence of labor with or without
previous cesarean section (Fig. 1C).
Expression of PGES in myometrium from pregnant women
Staining for PGES was localized in the cytoplasm of
smooth muscle myocytes (Fig. 2A). Ir-PGES was also observed in vascular smooth muscle cells and endothelial cells
of the blood vessels (Fig. 2B). Ir-PGES immunostaining was
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Giannoulias et al. • PGES and PGIS in Myometrium
FIG. 3. Dual immunofluorescent staining of PGES, PGIS, and ␣-actin in human myometrium. A, ␣-Actin labeled with Alexa Fluor 488. B, PGES
labeled with Alexa Fluor 568. C, The superimposed image of ␣-actin and PGES. D, PGIS labeled with Alexa Fluor 488. E, PGES labeled with
Alexa Fluor 568. F, The superimposed image of PGIS and PGES. Alexa Fluor 488 produces a green fluorescence. Alexa Fluor 568 produces a
red fluorescence. Yellow labeling represents colocalization. EN, Endothelial cells. All photographs taken at a magnification of ⫻200. Scale bar,
25 ␮m.
removed after preabsorption of the PGES antibody with the
PGES antigen (Fig. 2C). The localization pattern of PGES and
PGIS by immunohistochemistry was confirmed with dual
immunofluorescence. PGES colocalized with ␣-actin in
smooth muscle myocytes and was also present in the endothelium of blood vessels that did not immunostain for ␣-actin
(Fig. 3C).
PGES mRNA was detected by in situ hybridization in all
tissues examined. No signal above background was found
when tissues were incubated with the sense probe (Fig. 4, A
and B). There was no significant change in PGES mRNA with
advancing gestation or with labor at preterm and term (Fig.
4B). Western blot analysis of PGES identified two bands at
molecular masses of 180 and 16 kDa (Fig. 5A). The 16-kDa
band has been shown previously to be the active form of the
enzyme (6). There was no significant effect of gestational age
Giannoulias et al. • PGES and PGIS in Myometrium
FIG. 4. In situ hybridization of PGES in human myometrium. A,
Representative in situ of PGES term labor (antisense) and sense
control. B, The histogram represents the relative OD of PGES mRNA.
Data were analyzed by two-way ANOVA (␣ ⫽ 0.05). PT/NL, Preterm
no labor (n ⫽ 4); PT/L, preterm labor (n ⫽ 3); T/NL, term no labor (n ⫽
5); T/L, term labor (n ⫽ 5). All values are the mean ⫾ SEM.
on PGES protein levels of either the 180- or 16-kDa band (P ⬎
0.05; Fig. 5B), nor was there any significant change in ir-PGES
protein levels with labor at preterm or at term.
In human myometrium, we observed both 180- and 16kDa bands for PGES protein; however, the 180-kDa band was
predominant. We compared the expression of PGES in myometrium to that in placenta, which has high PGES activity (6).
In human placenta the PGES antibody identified two bands
at 180 and 16 kDa, but the 16-kDa band was predominant
(Fig. 6A). There was significantly more 16-kDa PGES protein
in human placenta (with or without labor) than in myometrium from pregnant women (with or without labor; Fig. 6B).
There was no significant difference in the 180-kDa band in
placenta compared with myometrium, with or without labor
(data not shown).
Expression of PGIS in myometrium from pregnant women
Ir-PGIS was localized to smooth muscle myocytes in myometrium from all four patient groups and also to vascular
smooth muscle cells. There was only very weak staining in
the endothelial cells (Fig. 2, D and E). Dual immunofluorescence staining indicated that PGIS colocalized with PGES in
smooth muscle cells (Fig. 3E). PGIS immunostaining was
abolished after preabsorption of the PGIS antibody with its
corresponding antigen (Fig. 2F).
PGIS mRNA was detected in myometrium from all four
J Clin Endocrinol Metab, November 2002, 87(11):5274 –5282 5279
FIG. 5. Western blot analysis of PGES protein in human myometrium. A, The first four lanes represent a Western blot of PGES
protein (180 and 16 kDa). The next four lanes represent the same
samples that were preabsorbed with the primary antibody. G␤ (40
kDa) was used as an internal control for protein loading. B, The
histogram represents the relative OD of PGES protein (18 kDa isoform) normalized to G␤ to control for protein loading. Data were
analyzed by two-way ANOVA (␣ ⫽ 0.05). PT/NL, Preterm no labor
(n ⫽ 8); PT/L, preterm labor (n ⫽ 4); T/NL, term no labor (n ⫽ 8); T/L,
term labor (n ⫽ 6). All values are the mean ⫾ SEM.
patient groups, and no signal above background was detected with the sense probe (Fig. 7A). There was a significant
decrease in PGIS mRNA levels at term compared with preterm patients (P ⬍ 0.05; Fig. 7B). However, there was no
significant change in PGIS mRNA with labor either preterm
or at term (Fig. 7C). Immunodetection of PGIS by Western
blot resulted in a single band with a molecular mass of 56 kDa
(Fig. 8A). There was a significant decrease in ir-PGIS protein
at term compared with preterm patients (P ⬍ 0.05; Fig. 8B).
However, there was no significant change in ir-PGIS protein
levels with preterm or term labor (P ⬎ 0.05; Fig. 8C).
Discussion
PGES and PGIS mRNA and protein were expressed in
myometrium from pregnant women throughout gestation,
suggesting that local production of PGE2 and PGI2 might be
important in the regulation of myometrial function. The
PGES and PGIS proteins were localized to the smooth muscle
myocytes in myometrium and smooth muscle cells of the
vasculature. There was no significant change in PGES and
PGIS protein levels with labor preterm or at term. However,
there was a significant decrease in PGIS mRNA and protein
with increasing gestational age consistent with a decreasing
influence of PGI2 on the myometrium in later gestation.
PGE synthase converts PGH2, the cyclic endoperoxide in-
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Giannoulias et al. • PGES and PGIS in Myometrium
FIG. 7. In situ hybridization of PGIS in human myometrium. A, Representative in situ of PGIS preterm no labor (antisense) and sense
control. B, Pooled all preterm patients vs. term patients. *, P ⫽ 0.04.
C, PT/NL, Preterm no labor (n ⫽ 4); PT/L, preterm labor (n ⫽ 3); T/NL,
term no labor (n ⫽ 5). All values are the mean ⫾ SEM. Data were
analyzed by two-way ANOVA (␣ ⫽ 0.05).
FIG. 6. Western blot analysis of PGES protein in term placenta and
human myometrium (Myo) during cesarean section (labor and no
labor). A, The first four lanes show a Western blot of PGES protein
in human myometrium (180 and 16 kDa) at term no labor. The next
five lanes represent PGES protein in human placenta (term no labor).
G␤ (40 kDa) was used as an internal control for protein loading. B, The
histogram represents the relative OD of PGES protein (the 16-kDa
isoform) normalized to G␤ to control for protein loading. Data were
analyzed by two-way ANOVA. *, P ⬍ 0.05, placenta vs. myometrium
T/NL, Myometrium term no labor (n ⫽ 5); T/L, myometrium term
labor (n ⫽ 3); T/NL, placenta (n ⫽ 4); T/L, placenta (n ⫽ 5). All values
are the mean ⫾ SEM.
termediate of PG synthesis, to PGE2. Likewise, prostacyclin
synthase converts PGH2 to PGI2. In the present study the
antibody we used recognized the membrane-bound form of
the PGES enzyme. In human cell lines mPGES is colocalized
with both PGHS enzymes in the perinuclear envelope (15).
We localized mPGES to the smooth muscle in the myometrium, similar to the localization pattern of PGHS-II as demonstrated previously (23) and consistent with studies that
have shown functional coupling of mPGES with either of the
PGHS isoforms (15). These results suggest that localization
of mPGES and PGHS-II to the smooth muscle of the myometrium may allow for efficient conversion of arachidonic
acid to PGE2 at that locale. Previously it had been demonstrated that human vascular smooth muscle cells, but not
endothelial cells, expressed PGES (27). However, we localized PGES to the smooth muscle cells and to endothelial cells
of the vasculature, consistent with a local effect of PGE2 on
vascular function. These differences may reflect different
blood vessels since Soler et al. (27) scanned sections of human
umbilical vein.
Western blot analysis of ir-PGES revealed immunoactive
protein bands of 180 and 16 kDa. The cytosolic form of PGES
immunoprecipitates as a protein of 23 kDa as described pre-
viously (14) and was not measured in this study. Previous
results have demonstrated that purified mPGES immunoprecipitated as two protein bands with molecular masses of
17 and 180 kDa, respectively (28). The 17-kDa protein had a
Km for PGH2 of 40 ␮m, and the larger protein had a Km of 150
␮m. PGH2 is therefore a better substrate for the smaller molecular mass PGES protein, suggesting that the smaller protein is probably the active protein (6, 28). We found no
significant change in the levels of PGES mRNA or protein
with increasing gestation or with labor at preterm or term.
No change in mPGES protein was observed in human amnion and chorion tissue between preterm or term patient
groups with or without labor (17). Therefore, changes in the
levels of this protein within intrauterine tissues do not appear to contribute to the marked increase in PGE2 output
from intrauterine tissues at the time of labor. Our results also
showed that whereas the 180-kDa band is the predominant
form expressed in myometrium from pregnant women, the
16-kDa band is the predominant band expressed in human
placenta and was present at much higher levels in the placenta. If this distribution reflects active enzyme, then the
production of PGE2 may be more important in placenta than
myometrium, perhaps signifying the importance of PGE2
regulating placental blood flow throughout pregnancy.
However, the action of PGE2 also depends on the receptor
subtypes present in both placenta and myometrium, and
future studies will need to evaluate the distribution and
changes in these receptor proteins.
Immunostaining and dual immunofluorescence of PGES
and PGIS suggested that these enzymes were associated with
smooth muscle myocytes, consistent with previous results
(23) showing PGHS and PGIS immunostaining in the same
cells. In our present study we localized PGIS in myocytes and
Giannoulias et al. • PGES and PGIS in Myometrium
FIG. 8. Western blot analysis of PGIS protein in human myometrium.
A, Western blot of PGIS protein. G␤ (40 kDa) was used as an internal
control for protein loading. B, PGIS protein, pooled all preterm patients
vs. term patients. *, P ⬍ 0.05. The histogram represents the relative OD
of PGIS protein normalized to G␤ to control for protein loading. Data
were analyzed by two-way ANOVA (␣ ⫽ 0.05). C, PT/NL, Preterm no
labor (n ⫽ 8); PT/L, preterm labor (n ⫽ 4); T/NL, term no labor (n ⫽ 8);
T/L, term labor (n ⫽ 6). All values are the mean ⫾ SEM.
smooth muscle cells in the vasculature, suggesting that the
high concentration of PGIS in uterine smooth muscle cells
could represent substantial PGI2 biosynthetic capacity. This
is consistent with reports that PGI2 is the main arachidonic
acid metabolite in the human myometrium (9), is produced
by the smooth muscle myocytes (19, 21), and contributes to
smooth muscle relaxation in the lower segment. In addition,
we colocalized PGIS with PGES in the same cell types, suggesting that these two enzymes are present in the same cell
acting to efficiently convert PGH2 into either PGE2 or PGI2.
Although the capacity to produce PGI2 appears to be an
inherent activity of the myocytes, the vasculature of the myometrium is also capable of PGI2 production (29). However,
immunostaining for PGIS in the smooth muscle of the vasculature was much less than that in myometrial cells, which
concurs with the findings of a previous study (21).
Western blot analysis of ir-PGIS in myometrium from
pregnant women revealed a single band at 56 kDa, which is
consistent with previous studies (19, 30, 31). We found no
significant change in PGIS protein or mRNA with labor preterm and at term. However, we did find a significant decrease in PGIS mRNA and protein with increasing gestational age, suggesting that PGI2 production may be more
important before term, when the myometrium is relatively
quiescent, than at term, when the myometrium switches into
a relatively contractile state. We appreciate that we have
taken lower segment tissues, and there could have been
J Clin Endocrinol Metab, November 2002, 87(11):5274 –5282 5281
variability in the amount of smooth muscle present from one
patient to another, especially those patients who had had a
previous cesarean section. However, after quantifying the
␣-actin content, we found no significant change in ␣-actin
protein during preterm or term labor or in patients who had
undergone a previous cesarean section. Our results agree
with those of Word et al., (32), who also found no significant
change in the actin content between myometrium of nonpregnant and pregnant women. Thus, we submit that the
lack of change in mPGES in different patient groups is not a
function of altered numbers of myocytes. Furthermore, the
different pattern of change in PGES and PGIS, both of which
localize predominantly to myocytes, indicates an independent pattern of regulation.
Levels of PGES and PGIS protein may not necessarily
reflect enzyme activity. Furthermore, the effects of PGE2 and
PGI2 may be more dependent on the receptor subtypes expressed in human myometrium. Senior et al. (33, 34) showed
that lower uterine segment tissue had more pronounced
responses to prostaglandin E2 receptor type 2 and inducing
protein receptor activation, consistent with the thesis that the
lower segment myometrium remains relatively passive at the
time of labor and dilates to allow passage of the fetus. Interestingly, PGE2 induced contraction of strips of myometrium obtained from the upper segment during labor,
whereas the lower uterine segment responded with inhibition (33). Lye and Challis (35) showed that in nonpregnant
sheep, the infusion of PGI2 significantly reduced the frequency and amplitude of uterine contractions. Similarly, in
myometrium from pregnant women, PGI2 significantly lowered the tonus and reduced spontaneous motility (10, 33).
Thus, our results are consistent with local production of PGI2
in myometrium, which may play a major role in sustaining
uterine quiescence during pregnancy, an effect that may be
diminished at the time of labor.
In conclusion, the presence of PGES and PGIS in human
myometrium supports a potentially important role for locally produced PGI2 and PGE2 in the regulation of myometrial activity. The expression of PGES did not change with
gestational age or the presence of labor, perhaps indicating
the greater importance of alterations in PGE receptor subtypes in effecting PGE2 action. However, the significant decline in levels of PGIS mRNA and protein in later gestation
suggest that local withdrawal of this inhibitory PG in the
myometrium may contribute to the labor process.
Acknowledgments
We thank Ljiana Petkovic, Cristine Botsford, and Lindsay McWhirter
(Mount Sinai Hospital, Toronto, Ontario, Canada) for their assistance
with collecting tissues for these studies, and Nohjin Kee for help with
the dual immunofluorescent studies.
Received April 2, 2002. Accepted August 5, 2002.
Address all correspondence and requests for reprints to: Ms. Diana
Giannoulias, 1 King’s College Circle, Medical Sciences Building, Room
3344, Department of Physiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8. E-mail address: [email protected].
This work was supported by the Canadian Institutes of Health Research in Human Development, Child and Youth Health (MOP 42378).
5282 J Clin Endocrinol Metab, November 2002, 87(11):5274 –5282
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