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Original Studies |
Sir Quinton Hazell Molecular Medicine Research Center, Department of Biological Sciences, University of Warwick, Coventry, United Kingdom CV4 7AL
Address all correspondence and requests for reprints to: Dr. D. Grammatopoulos, Sir Quinton Hazell Molecular Medicine Research Center, Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry, United Kingdom CV4 7AL. E-mail: chdg{at}dna.bio.warwick.ac.uk
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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, -R1ß, -R1c, and -R2ß) to
investigate the possible regulation of PG production by CRH. We studied
the effect of CRH on the two major myometrial PGs, PGE2 and
6-keto PGF1. Human CRH was able to partially inhibit
basal, interleukin-1ß-stimulated, and oxytocin-stimulated
PGE2 production (56 ± 11%, 45 ± 8%, and
58 ± 6% inhibition, respectively). This effect was blocked by a
specific CRH receptor antagonist in a concentration-dependent manner.
Furthermore, CRH had no effect on 6-keto PGF1
production, indicating that the CRH inhibitory action does not involve
suppression of cyclooxygenase, the enzyme responsible for the
production of both PGE2 and 6-keto-PGF1.
These data further support the view that during pregnancy, CRH may
promote myometrial quiescence and might play an important role in the
regulation of human labor. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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subunits (12) and 2) an inhibitory action of oxytocin, which activates
protein kinase C, leading to phosphorylation and desensitization of the
CRH receptor (13).
Several reports indicate that CRH exerts differential actions on PG
production depending on the cell type. It stimulates PG production in
placenta, decidua, and fetal membranes in vitro (14) and
inhibits interleukin-1 (IL-1)-induced 6-keto-PGF1
release from bovine aorta endothelial cells and PGE2
release from human dermal fibroblasts (15). Although it has not been
demonstrated unequivocally, PGs appear to play in important role in the
mechanism of labor, and increased PG production is associated with term
and preterm labor, especially in the presence of intrauterine infection
(16, 17).
The fetal membranes, especially the amnion, produce predominantly PGE2, which appears to be important in the early stages of labor, leading to cervical ripening and dilatation. It has been suggested that increased PG synthesis during labor is the result of increased transcription of the cyclooxygenase-2 (COX-2) gene, which is the inducible form of the enzyme (18). In humans, COX-2, but not COX-1 (the constitutive form), expression and activity increase at term before the onset of labor in amnion (19), chorion (20), and myometrium (21), but not in decidua (22). Furthermore, although myometrial COX-1 expression was unaffected in labor, COX-2 expression was reduced in labor in both preterm and term pregnancies (21). Interestingly, in mice with targeted disruption of the COX-1 gene, the process of labor was delayed, resulting in neonatal death (23), whereas the targeted disruption of COX-2 produced multiple failures in female reproductive processes, including ovulation, fertilization, implantation, and decidualization (24). In a range of cell types, including human amnion (25), decidua (26), and myometrium (27), the production of PGs appears to be part of an auto/paracrine system involving various cytokines, and this has been implicated in the initiation of infection-induced preterm human labor (28).
To investigate further the role of CRH during pregnancy and labor and elucidate in detail its actions in the human myometrium, we have studied its possible involvement in the regulation of PG production from primary human pregnant myometrial cell cultures that express functional CRH receptors.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Pregnant myometrial tissue was obtained from women undergoing elective caesarean section for nonmaternal reasons at term before the onset of labor (n = 9). The biopsy was standardized to the upper margin of the lower segment of the uterus in the midline. This provides the closest approximation of the upper segment of the uterus. The patients had no clinical evidence of intrauterine infection. The tissue was immediately processed for cell culture. Ethical approval was obtained from the local ethical committee, and each patient gave informed consent to the study.
Chemicals
Human/rat CRH, -helical CRH-(9–41), and oxytocin were
obtained from Peninsula Laboratories (Merseyside, UK). PGE2
and 6-keto-PGF1 RIA kits and Amprep
C18, C2 minicolumns were obtained from
Amersham International (Little Chalfont, UK). Mouse
monoclonal vimentin antibody and anti-mouse IgG tetramethyl-rodamine
isothiocyanate (TRITC)-conjugated were obtained from Sigma Chemical Co. (Poole, UK). RNeasy plant total ribonucleic acid
(RNA) kit for polyadenylated RNA isolation was obtained from
QIAGEN (Crawley, UK) Mouse monoclonal muscle actin
antibody was obtained from DAKO Corp. (High Wycombe, UK).
Synthetic oligonucleotide probes and enzymes were purchased from
Life Technologies (Paisley, UK). Antigoat IgG-fluorescein
isothiocyanate-conjugated and specific CRH receptor antibody (which
recognizes both human CRH-R1 and CRH-R2 receptors) were obtained from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The
latter is a goat polyclonal antibody raised against a peptide
corresponding to amino acids 425–444 mapping at the C-terminus of the
human CRH-R1 precursor. All other chemicals were purchased from
Sigma Chemical Co. (Poole, UK). cAMP RIA kits were
obtained from New England Nuclear Life Science (Boston, USA). IL-1ß
(recombinant) was obtained from Calbiochem (Nottingham,
UK)
Preparation of Myometrial Cell Cultures
Myocytes were prepared by enzymatic dispersion as previously
described (13). Briefly, pieces of myometrium were transferred into
DMEM containing collagenase (300 U/mL), deoxyribonuclease (30 U/mL),
penicillin (200 U/mL), and streptomycin (200 mg/mL) and incubated at 37
C for 30 min. After filtration and centrifugation, cells were suspended
in DMEM containing 10% FCS, penicillin (100 U/mL), streptomycin (100
mg/mL), and fungizone (2.5 µg/mL). The cells were kept at 37 C in a
humidified atmosphere of 95% air and 5% CO2 until
confluent (
2 weeks). The purity of myometrial muscle cells was
assessed by immunocytochemical staining. Mouse antihuman smooth muscle
actin-specific monoclonal antibody and peroxidase-conjugated rabbit
antimouse antibody were used. Human fibroblast cells and omission of
the primary antibody were used as negative controls, whereas frozen
myometrial tissue was used as a positive control. To minimize
fibroblast contamination we repurified the myocyte preparation 48
h before the experiments using 0.5% trypsin.
RNA extraction and RT-PCR
To investigate the CRH receptor subtypes present in the human
pregnant myometrial cells, a RT-PCR technique was used. Different
specific primers were used for the CRH-R1 subtypes as previously
described (7), whereas for the R2 subtypes the following primers were
used. For CRH-R2ß, primers 5'-TCCAGTCCCTAACCCCAGCC-3' and
5'-GGGAATTCCCGGGCCAAGAGGCATGGTTTATTTC-3' were used for the first round,
and primers 5'-CTGGCATGAGGGGTCCCTCAG-3' and 5'-GGGAATTCCCGGGGCA
GGTGGGCGACCGAGGG-3' were used for the second round of PCR. For
CRH-R2
, primers 5'-CTGTGCTCAAGCAATCTGCCTAC-3' and
5'-GGGAATTCCCGGGCCAAGA GGCATGGTTTATTTC-3' were used for the first
round, and primers 5'-CTTGGCTTCCCCAAGTGCTGAG-3' and
5'-GGGAATTCCCGGGGCAGGTGGGCGA CCGAGGG-3' were used for the second round
of PCR. The products of the second PCR reaction were confirmed using
sequence analysis.
Immunofluorescence
Fixed myometrial cells grown on glass slide-flaskettes, washed in phosphate-buffered saline (PBS), and incubated with 3% BSA for 1 h (to block nonspecific binding sites) before incubation with the primary CRH-R antibody for 60 min, which was used at a 1:100 dilution (all dilutions were made in 3% BSA in PBS). After three washes with PBS, specimens were incubated for 30 min with a primary mouse monoclonal actin antibody (1:100) followed by another set of washes as before. Incubation with the first secondary antigoat IgG-fluorescein isothiocyanate-conjugated antibody was carried out for 2 h in the dark, followed by three washes with PBS and addition of the second secondary antimouse IgG tetramethyl-rodamine isothiocyanate (TRITC)-conjugated antibody for 30 min. Specimens were washed thoroughly, and the coverslips were mounted using 90% glycerol-PBS. The results were viewed under fluorescent microscope using appropriate filters.
cAMP production assay and RIA
Human myometrial cells from pregnant women (pregnant myometrial cells) were plated in 24-well plates at an average density of 5 x 104 cells/plate and after reaching confluence were incubated in 0.2 mL 10% FCS in DMEM containing 20 mmol/L HEPES, 500 µmol/L isobutylmethylxanthine (pH 7.2), and suitable concentrations of human/rat CRH for 10 min at 37 C. Experiments were terminated by the addition of 0.1 mL 0.3 mol/L HCl. Cells were frozen overnight, and cAMP levels were measured in the supernatants using RIA. The sensitivity of the assay was 0.025 pmol, and the precision was: intraassay coefficient of variation (CV), 2.9%; and interassay CV, 9.7%.
PG stimulation assay: determination of 6-keto-PGF1a and PGE2
Human pregnant myometrial cells were plated in 12-well plates at
an average density of 5 x 106 cells/plate and after
reaching confluence were incubated in 2 mL 10% FCS in DMEM containing
suitable concentrations of the agents tested (CRH, IL-1ß, -helical
CRH, and oxytocin) for the time indicated in each experiment.
Experiments were terminated by transferring the medium to test tubes.
Immediately both PGs were extracted from samples according to the
following methods.
PGE2. One milliliter of samples were mixed with 1 mL water-ethanol (1:4) and 20 µL glacial acetic acid, vortexed, and left at room temperature for 5 min. After centrifugation at 2500 x g for 2 min, supernatants were applied to Amprep C18 minicolumns that had been primed with 2 mL 10% ethanol. The columns were washed with 1 mL water and 1 mL hexane. PGE2 was eluted with 2 x 0.8 mL ethyl acetate and evaporated to dryness under vacuum overnight. Just before the RIA, samples were reconstituted with 100 µL PBS, pH 7, containing 0.1% gelatin. Using this method, the recovery of radiolabeled PGE2 was 91 ± 4%.
6-Keto-PGF1|ga. One milliliter of
samples was acidified with 1 mol/L citric acid and applied to Amprep
C2 minicolumns that had been primed with 2 mL methanol
and 2 mL water. The columns were washed with 5 mL water, 5 mL 10%
ethanol, and 5 mL hexane. 6-Keto-PGF1 was eluted with 5
mL methyl formate, which was then evaporated to dryness under vacuum
overnight. Just before the RIA, samples were reconstituted with 1 mL
RIA assay buffer. Using this method, the recovery of radiolabeled
6-keto-PGF1 was 88 ± 3%. PGs were measured by
RIA. For the PGE2 assay, antiserum against the methyl
oximate derivative was used, which has cross-reactivity with other PGs
as follows: PGE1, less than 5%; and all other PGs
[8-iso-PGE2, 19(OH)-PGE2,
15-keto-PGE2, 6-keto-PGE1, PGD2,
PGF2, 6-keto-PGF1], less than 0.01%.
The sensitivity of the assay was 1 pg/tube, and the precision was:
intraassay CV, 8.9%; and interassay CV, 11%. For the
6-keto-PGF1 assay, antiserum against
6-keto-PGF1 was used, which has cross-reactivity with
other PGs as follows: PGF1, 1%; PGF2,
0.8%; PGE1/E2, 0.5%; and thromboxane
B2, less than 0.3%. The sensitivity of the assay was 3
pg/tube, and the precision was: intraassay CV, 9.6%; and interassay
CV, 13.2%.
Statistical analysis
Data are shown as the mean ± SEM of each measurement. Data were tested for homogeneity, and comparison between group means was performed by one- or two-way ANOVA. P < 0.05 was considered significant.
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Results |
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Using immunofluorescence with a CRH-R1/2-specific antibody, a
plasma membrane rich in immunopositive granules was revealed (Fig. 1A
). To confirm the type of cells present
in the cultures that were immunopositive for the receptor, double
staining was performed on the same specimens for CRH-R1/2 and actin
(specific smooth muscle cell marker). As shown in Fig. 1B, the cells
were positive for actin, suggesting that our primary cultured human
pregnant myometrial smooth muscle cells express specific membrane-bound
CRH receptors. In preliminary experiments, HEK293 cells stably
transfected with CRH-R1. CRH-R1ß or CRH-R2ß receptors were used
as positive controls, whereas untransfected 293 cells were used as
negative controls (data not shown). Omission of the anti-CRH receptor
antibody was also used as a negative control (data not shown).
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, CRH-R1ß,
CRH-R1c, and CRH-R2ß (Fig. 2a). No
CRH-R2 messenger RNA (mRNA) was identified, consistent with the idea
that this receptor is confined to the brain only. Interestingly,
CRH-R2 mRNA was not found in these cell cultures, a receptor subtype
that was present in biopsies from pregnant, but not nonpregnant,
myometrium. Because RNA was isolated from the cells on the day of
confluence (on day 13 ± 2), we investigated the effect of the
culture period on the CRH-R subtype profile. Identical CRH-R subtypes
were found in the cells from the 5th day up to the 10th week of culture
(4th passage).
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). CRH caused a
dose-dependent increase in adenylate cyclase activity, with a threshold
of 50–100 pmol/L CRH and a maximal increase (350 ± 76% increase
in basal) at a concentration of 100 nmol/L. No significant variation
was observed between different preparations of myometrial cells. Effect of IL-1ß on PG production
Exposure of cultured human pregnant myometrial cells to IL-1ß in
a concentration range of 1–100 ng/mL stimulated the release of
PGE2 and PGI2, as measured by its stable
metabolite 6-keto-PGF1; in contrast, IL-1ß in a
concentration range of 0.01–0.5 ng/mL had no effect (data not shown).
The concentration of IL-1ß that produced maximal PG release was 10
ng/mL and was used in subsequent experiments. No significant variation
was observed between the preparations of myometrial cells tested
(n = 9). The IL-1ß-stimulated release of PGs was also time
dependent (Fig. 3). IL-1ß was able to
stimulate a significant increase in PGs after 8–12 h of incubation,
achieving maximal responses after 18 h (200 ± 45% above
basal levels). In time-matched untreated controls, PG production was
also time dependent, and the variation in PG production over time was
24 ± 5%. Furthermore, in our culture system PGE2 was
the major PG released, with 6-keto-PGF1 concentrations
being 4 times less than those of PGE2 (Table 1).
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In human myometrial cells, CRH was able to inhibit both basal and
IL-1ß-stimulated PGE2 production (56 ± 11%
inhibition of basal and 45 ± 8% inhibition of
IL-1ß-stimulated; Fig. 4a). In
contrast, CRH had no effect on basal or IL-1ß-stimulated levels
of 6-keto-PGF1 production (Fig. 4b). In subsequent
experiments it was found that the CRH effect on PGE2
production was rapid, reaching maximal inhibition within the first 30
min of incubation, and was persistent for up to 18 h (Fig. 5a). It was also found to be
concentration dependent, with maximum inhibition observed at a CRH
concentration range of 1–10 nmol/L (Fig. 5b). Interestingly, at high
concentrations (100 nmol/L), CRH was able to induce a small, but
significant (22 ± 7%), increase in PGE2
production.
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-helical CRH-(9–41) in a
concentration range of 100 nmol/L-1 µmol/L was coincubated with CRH
(Fig. 6).
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Oxytocin in a concentration range of 0.1–10 nmol/L was able to
stimulate PGE2 release in human pregnant myometrial cells
(data not shown). PGE2 levels were increased by 200 ±
30% above basal after 30-min incubation with 10 nmol/L oxytocin (Fig. 7). When human CRH (10 nmol/L) was
coincubated with oxytocin (10 nmol/L), no significant increase in basal
PGE2 levels was observed.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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, -R1ß, -R1c, and -R2ß. We also
showed that these receptors are functional, as upon stimulation with
CRH, they can activate adenylate cyclase and cAMP production,
consistent with previous observations (9). However, at present and
because of the lack of receptor subtype-specific antibodies, it is not
possible to identify the relative receptor subtype expression.
Furthermore, these cell cultures do not contain mRNA for CRH-R2,
although the tissues of origin (pregnant myometrial tissue obtained
from women at term undergoing elective caesarean section) were found to
be positive for that particular CRH-R subtype mRNA. This finding
suggests that maternal factors that are components of the pregnant
environment may play a major role in the regulation of this subtype
expression. However, only studies on the CRH-R2 promoter will help to
elucidate in detail this phenomenon. These findings indicate the
limitations of experiments performed in cells cultured for long periods
of time and the difficulties present in extrapolating and interpreting
the results from these studies to the in vivo phenomena. One
explanation is that our cultures contain predominantly smooth muscle
cells during the culture period, and the type 2 receptor in
vivo may be expressed in another cell type, such as uterine
fibroblasts. Using immunofluorescence in pregnant myometrium tissue
sections, we have previously reported that CRH receptors (R1 and/or R2)
are present in uterine fibroblasts (7). This phenomenon is currently
under investigation. In this study we restricted our experiments to
relatively pure cultures of myocytes to enable the data to be
interpreted more easily.
Consistent with previous reports (28), IL-1ß was able induce the
secretion of PGE2 and PGI2, as measured by its
stable metabolite 6-keto-PGF1, from myometrial cells in
a time- and concentration-dependent manner. We studied the effect of
CRH on these two major myometrial PGs. Interestingly, the myometrium is
the only tissue from the feto-maternal membranes that has the capacity
to secrete appreciable amounts of 6-keto-PGF1 (30).
Although CRH at physiological concentrations had no effect on basal or
IL-1ß-stimulated 6-keto-PGF1, it exerted a complex
effect on basal and IL-1ß-stimulated PGE2 production. In
a CRH concentration range of 1–10 nmol/L, which is around the
physiological concentration of CRH at term, it was able to suppress
both basal and IL-1ß-stimulated PGE2 production. This is
a novel finding, which indicates that CRH may play a role in the
regulation of myometrial quiescence. It is well known that a number of
enzymes are involved in PG biosynthesis from the principal precursor
arachidonic acid, COX, which converts arachidonic acid into cyclic
endoperoxide (PGG2), which, in turn, is converted rapidly
into PGH2 by the action of peroxidase. It is now thought
that the same protein is responsible for both reactions and is called
PG endoperoxide H synthetase (COX). These intermediates can be further
metabolized to PGs and thromboxanes by the actions of various
isomerases. PGI2 is derived by the action of prostacyclin
isomerase, whereas PGE2 is the result of the action of
endoperoxide isomerase on PGH2. Our data suggest that the
action of CRH on myometrial cells is not targeted on the COX or
prostacyclin isomerase expression/activity, as the PGI2
pathway remains unaffected by CRH action. It is possible that CRH is
able to diminish the activity or expression of endoperoxide isomerase,
which is the only enzyme responsible for PGE2 generation.
Another possibility is that CRH might influence the rate of
PGE2 metabolism to PGF2,
13,14-dihydro-15-oxo-PGF2, or
13,14-dihydro-15-oxo-PGE2 by modulating the corresponding
enzymes, such as the PG-9-oxo-reductase, that are present in the
myometrium (35).
The exact intracellular signaling pathway by which IL-1ß can stimulate PG release in human myometrial cells is not yet fully understood. Stimulation of high affinity IL-1 receptors leads to increased levels of cAMP, possibly by the induction of one or more of the components of the adenylate cyclase system (31). This action of IL-1ß on PG production could be potentiated by protein kinase C activation (32). Also, in amnion the stimulatory action of IL-1ß on PGE2 production is attenuated by down-regulation of protein kinase C (33). In contrast, in human decidual cells basal PGE2 production is stimulated by protein kinase C activation, whereas IL-1ß-stimulated PGE2 synthesis is not affected by down-regulation of this enzyme, suggesting that IL-1ß and protein kinase C stimulate PGE2 production via different mechanisms (34).
At present, the exact pathway or receptor subtype by which CRH partially inhibits PGE2 release is not known. It is possible that activation of the adenylate cyclase-cAMP-dependent protein kinase is involved, as we have shown that CRH can activate this pathway in human myometrial cells. It has been demonstrated that in amnion, activation of this enzyme attenuates the stimulatory effect of epidermal growth factor (36) and oxytocin (37) on PGE2 production, whereas in human chorion and decidual cells, activation of adenylate cyclase results in enhanced stimulatory action of IL-1ß, epidermal growth factor, and phorbol 12-myristate 13-acetate on PG production (38). These observations suggest that activation of the adenylate cyclase-cAMP-dependent protein kinase pathway can have effects on PG production that are specific with respect to tissue source and presence of other activators/inhibitors.
Interestingly, at higher concentrations CRH induced a small, but significant, stimulation of PGE2 release. These data are in agreement with our previous observations, where we described a small, but significant, stimulatory effect (20 ± 6% increase from basal) of CRH on PGE2 production from membranes prepared from human myometrial biopsies at term (9). Although this might represent a pure pharmacological effect, it is also possible that within the myometrial cells different CRH-R subtypes mediate diverse and complex cellular responses and actions, such as activation of phospholipase A2 activation, which leads to increased arachidonic acid production and increased PGE2 biosynthesis. However, specific CRH-R subtype antagonists will be needed to test this hypothesis.
We have also shown that in human pregnant myometrial cells oxytocin in a concentration range of 0.1–10 nmol/L is able to stimulate PGE2 release. It is well accepted that in the human myometrium oxytocin activates the phospholipase C-inositol triphosphate-protein kinase C pathway (39, 13), and this action of oxytocin on myometrial PGE2 release is consistent with the hypothesis that in some intrauterine tissues, such as the amnion, activation of protein kinase C can stimulate PGE2 production (40). The observation that CRH is able to inhibit the oxytocin-induced PGE2 stimulation suggests that at term before the onset of human labor CRH might act as a signal that exerts a general inhibitory effect on PGE2 production independent of which pathway is activated.
Although both stimulatory (via generation of PG from the fetal membranes) and inhibitory (via stimulation of adenylate cyclase) actions of myometrial contractility have been proposed (41, 42, 43), the role of CRH during pregnancy remains unknown. The presence of multiple CRH receptor subtypes in the human pregnant myometrium suggests distinct functional roles for each receptor during pregnancy and raises the possibility of multiple roles for CRH and/or related peptides. It is attractive to speculate that some CRH receptor isoforms may be responsible for the maintenance of myometrial quiescence via generation of cAMP and/or inhibition of myometrial PGE2 production. At term, inhibition of the biological activity of these receptor subtypes may enable CRH to exert different actions and therefore to play a central role in the control of uterine contractility and the mechanism of labor (13).
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Acknowledgments |
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Footnotes |
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Received December 15, 1998.
Revised February 17, 1999.
Revised March 4, 1999.
Accepted March 12, 1999.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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s in human myometrium
in term and preterm labor: A mechanism for parturition. J Clin
Endocrinol Metab. 79:1835–1839.[Abstract]
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