Departments of 1 Microbiology and Immunology, 2 Environmental Medicine, and 3 Pediatrics and Obstetrics and Gynecology, University of Rochester School of Medicine and Dentistry, Lung Biology and Disease Program, Rochester, New York 14642
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ABSTRACT |
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A
key role exists for prostaglandins (PGs) in reproductive health,
including fertility and parturition. However, the cellular sources and
regulation of PG production by cyclooxygenase (COX) in the human female
reproductive tract remain poorly understood. We recently reported that
human female reproductive tract fibroblasts are divisible into distinct
subsets based on their Thy-1 surface expression. Herein, we demonstrate
that the expression, induction, and subcellular localization of COX-1
and COX-2 and the downstream PG biosynthesis are markedly different
between these subsets. Specifically, Thy-1+ fibroblasts
highly express COX-1, which is responsible for high-level PGE2 production, a feature usually attributed to the COX-2
isoenzyme. In contrast, COX-2, generally considered an inducible
isoform, is constitutively expressed in the Thy-1 subset,
which only minimally produces PGE2. The intracellular signaling pathways for COX regulation also differ between the subsets.
Determination of differences in signal transduction, COX expression and
localization, and PG production by human reproductive fibroblast
subtypes supports the concept of fibroblast heterogeneity and the
possibility that these subsets may play unique roles in tissue
homeostasis and in inflammation.
inflammation; myometrium; lipid mediators; heterogeneity; prostaglandin; cyclooxygenase
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INTRODUCTION |
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CRITICAL EVENTS in the human female reproductive tract such as ovulation, implantation, and parturition are characterized by inflammation (9). Close interaction among the different cell layers that form the uterus, namely, the endometrium and myometrium, is required for the successful outcome of these events and is crucial for human existence. Besides cytokines and sex steroid hormones, prostaglandins (PGs) play a central role in the normal functioning of the reproductive tract in processes such as ovulation (7), menstruation (9, 18), implantation (24,) and parturition (25).
PGs are arachidonic acid metabolites, crucial for normal physiology and for inciting and regulating inflammatory responses. Prostanoid biosynthesis is catalyzed by two cyclooxygenases (COXs), COX-1 and COX-2. COX-1 is considered to be a constitutively expressed enzyme that maintains cellular homeostasis, whereas COX-2 is typically induced after stimulation with certain bacterial products and proinflammatory cytokines such as interleukin (IL)-1 and plays a major role in inflammation (40). The various types of PGs are ultimately synthesized by newly characterized synthases that may be differentially expressed among cells and tissues (16).
The study of COXs and PGs comprises a vast area of research in the biology of reproduction. PGs are essential for parturition in mice (12) and are crucial for initiating labor in humans (28, 44). A key PG, called PGE2, is crucial for inflammation, for reproductive tract functions, and for fetal organ maturation (33). COX expression is vital for "cervical ripening," a process of neutrophil tissue infiltration essential for normal labor (17). COX-1 has also been shown to be critical for normal labor in the mouse, because COX-1-deficient mice demonstrated delayed parturition, resulting in neonatal death (12). COX-1 and COX-2 mRNA is expressed in cow myometrium (11), and COX-1 and COX-2 protein exists at high levels in the human myometrium (41). Increased levels of COX-2 in the myometrium are associated with the onset of labor in women (8).
The concept of fibroblasts as "sentinel cells" has provided a new
role for them as initiators of inflammation and regulators of immunity
(5, 32, 37). Resident tissue fibroblasts produce proinflammatory cytokines and PGs upon stimulation and thus recruit and
communicate with classic immune cells. Fibroblasts themselves, however,
remain poorly understood and were previously considered to be only
structural cells with little intrinsic heterogeneity. An emerging
concept is that fibroblasts are not a homogeneous population but,
rather, consist of subsets that have organ-specific functions. For
example, fibroblasts are heterogeneous with respect to expression of
the surface antigen Thy-1 in several organs, including mouse lung
(31) and spleen (4), rat lung
(26), and human orbit (39). Little is known,
however, about fibroblasts derived from the human female reproductive
tract. We recently demonstrated that fibroblasts derived from human
myometrium are heterogeneous, and subsets separated on the basis of
Thy-1 expression have distinct cytokine and chemokine profiles
(21). In the current study, we investigated COX-1
and COX-2 expression by Thy-1+ and Thy-1
human myometrial fibroblasts. We demonstrate that myometrial fibroblast
subsets have distinct COX expression patterns, subcellular localization, and signal transduction pathways, as well as PG production. The findings in this report support the concept that human
myometrial fibroblast subpopulations contribute uniquely to
reproductive tract processes associated with inflammation. Our results
have important implications for the way in which reproductive tract
fibroblasts are viewed. In light of the evidence presented herein,
myometrial fibroblasts should now be viewed as key players in PG
homeostasis as well as in the initiation of inflammatory events in the
reproductive tract.
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MATERIALS AND METHODS |
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Tissue collection and fibroblast strain derivation. Tissue samples were obtained as previously described (19). In brief, biopsies were collected from myometrium in women undergoing gynecological procedures for benign conditions. All women had regular menstrual cycles (25-35 days), were premenopausal (20-42 y old), and did not receive any form of hormonal treatment during the 3-mo period before the procedure. Written informed consent was received from all patients, and ethical approval was obtained from the University of Rochester research subjects review board.
Tissue was cut into 1-mm3 pieces, and fibroblast cultures were established by standard explant techniques as previously described (10, 35). The cells were immunostained for fibroblast markers as previously described (19, 21). In brief, cells were positive for vimentin (fibroblast marker) but negative for cytokeratin (epithelial cell marker),RNA isolation and RNase protection assay.
Total RNA was isolated from myometrial fibroblast subsets at 0, 1, 2, 4, 6, 8, and 18 h after IL-1 treatment by using TRI-reagent (Molecular Research Center, Cincinnati, OH). RNA (8 µg) was used for
each sample to hybridize with the probe. COX-1 (human) antisense probe
template (nucleotides 379-751 of cDNA sequence accession no.
S78220; Cayman Chemical, Ann Arbor, MI), COX-2 (human) antisense probe
template (nucleotides 330-752 of cDNA sequence accession no.
M90100; Cayman Chemical), and a human
-actin antisense control
template (nucleotides 947-704 of cDNA sequence accession no.
X00351; Ambion, Austin, TX) were used to prepare labeled RNA probes
with the MAXIscript in vitro transcription kit (Ambion). The RNase
protection assay was performed by using the RPA II kit (Ambion)
according to the manufacturer's protocol.
Immunocytochemistry for COX-1 and COX-2.
Myometrial Thy-1+ and Thy-1 fibroblasts were
seeded in chamber slides (Nunc, Naperville, IL) in RPMI with 10% FBS.
Cells were serum-starved in 0.5% FBS for 72 h and then stimulated
with IL-1
(10 ng/ml; R&D Systems, Minneapolis, MN) or left untreated
for 24 h in serum-free medium. Cells were fixed with 2%
paraformaldehyde and then stained with a monoclonal mouse anti-human
COX-1 or monoclonal mouse anti-human COX-2 antibody (10 µg/ml; Cayman
Chemicals) or with isotype control mouse IgG1 or mouse IgG2b (Caltag,
Burlingame, CA). Biotinylated horse anti-mouse IgG (heavy and light
chain) (1:200; Vector Labs, Burlingame, CA) was added as a secondary antibody, and streptavidin-horseradish peroxidase (1:1000; Jackson ImmunoResearch Labs, West Grove, PA) was added as a substrate. Samples
were visualized by adding an aminoethyl-carbachol chromogen (Zymed,
South San Francisco, CA) and cover-slipped by using Immu-mount (Shandon, Pittsburgh, PA).
PG production.
Fibroblasts were treated as described above. PGE2
production was assayed by using an enzyme immunoassay (Cayman
Chemicals). At 2 h before the addition of IL-1, cultures were
treated with ethanol (vehicle control), indomethacin (10 µM; Sigma,
St. Louis, MO), SC-58125 (5 µM) or SC-58560 (0.5 µM). SC-58560 and
SC-58125 were kind gifts from Dr. Peter Isakson (Searle, Skokie, IL).
Western blot analysis.
Cells were treated as above for COX-1 and COX-2 analysis or treated
with IL-1 (10 ng/ml) and harvested in a time course at 1, 2, 5, 15, and 30 min, 1 h, and 6 h for extracellular signal-regulated kinase (ERK)1 (p42), ERK2 (p44), and p38 mitogen-activated
protein (MAP) kinase analysis. The ERK1/2 inhibitor PD-98059
(Calbiochem, San Diego, CA) inhibits MAP kinase/ERK kinase
(MEK1) activation and thus phosphorylation of ERK1/2 and was
added at 50 µM at the indicated time points or overnight. The p38
inhibitor SB-203580 (Calbiochem) was also added at 50 µM overnight.
DMSO was added as vehicle control to untreated cells. Equal amounts of
fibroblast protein lysates were subjected to Western blot analysis as
previously described (36). Mouse anti-human COX-1 and
mouse anti-human COX-2 antibodies were added in blocking buffer. The
phospho-p44/42 MAP kinase (Thr-202/Tyr-204) E10 monoclonal antibody in
mouse (1:2,000) and phospho-p38 MAP kinase (Thr-180/Tyr-182) antibody in rabbit (1:1,000) were purchased from Cell Signaling Technologies (Beverly, MA). Positive controls included phosphorylated p42 and p38
MAP kinase cell extracts (5 and 10 µl).
Statistical analysis. Statistical significance was determined by using Student's paired two-tailed t-test or a generalized linear model analysis, where P < 0.05 indicates statistical significance between the samples tested. Log transformation was performed to correct for variance.
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RESULTS |
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COX-1 and COX-2 mRNA expression by myometrial fibroblast subsets.
Fibroblasts are one of the main cell types responsible for PG
production via expression of COX-1 and COX-2 (5, 46). COXs are also central for homeostasis in the reproductive tract (17, 29). For these reasons, we first examined whether or not
Thy-1+ and Thy-1 myometrial fibroblasts
express COX-1 and/or COX-2 mRNA. Thy-1+ and
Thy-1
fibroblasts were left untreated or stimulated in a
time course with IL-1
, a cytokine abundant in the reproductive tract
and a known inducer of COX-2 (15). In particular, IL-1 is
present in the uterus throughout the menstrual cycle and during
pregnancy as a product of reproductive tract macrophages
(18). COX-1 and COX-2 mRNA levels were quantitated by
normalizing to
-actin by using an RNase protection assay.
Thy-1+ myometrial fibroblasts expressed COX-1 mRNA
constitutively, whereas COX-2 mRNA was absent in untreated cells but
was greatly upregulated after IL-1
treatment (10 ng/ml) (Fig.
1, A and B). COX-1
mRNA levels in myometrial Thy-1
fibroblasts were
relatively unchanged throughout the IL-1
time course. Interestingly,
constitutive COX-2 mRNA levels were detected in Thy-1
myometrial fibroblasts and, surprisingly, were not affected by IL-1
treatment (Fig. 1, C and D). This is an
intriguing finding because COX-2 is thought of as an inducible enzyme,
associated with inflammatory processes.
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COX-1 and COX-2 protein expression by Western blot.
Western blot analysis was next performed on untreated and
IL-1-stimulated (24 h) myometrial parental fibroblasts, as well as
on Thy-1+ and Thy-1
subsets (Fig.
2). A strain of human lung fibroblasts
treated with IL-1
was used as a strong positive control (lane
1) for both COX-1 and COX-2 (46). COX-1 is seen as a
68-kDa band on a Western blot. Our results demonstrate that COX-1
protein was highly expressed in both unstimulated and IL-1
-treated
Thy-1+ fibroblasts. The parental fibroblast strain showed
similar results but with less intense bands. COX-1 protein expression
in Thy-1
cells was barely detectable, seen as very faint
bands. COX-2 is typically observed in fibroblasts as a 72-kDa band.
Interestingly, the unstimulated myometrial parental fibroblasts
possessed a band of a mass somewhat smaller than 72 kDa, which remained
constant with IL-1
treatment. The typical 72-kDa COX-2 band was
expressed only after IL-1
stimulation. The isolated
Thy-1+ fibroblasts expressed the 72-kDa band, which was
absent in unstimulated cells but was upregulated after addition of
IL-1
. In contrast, the Thy-1
population displayed only
the smaller COX-2 band in both unstimulated and IL-1
-treated cells,
supporting the data showing constitutive mRNA for COX-2 in this
myometrial fibroblast subset (Fig. 1D). Interestingly, human
myometrial tissue expresses only the 70-kDa band for COX-2 (data not
shown).
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COX-1 and COX-2 expression in human myometrium.
In situ staining for COX-1 and COX-2 was performed on human myometrium
tissue sections. Immunohistochemistry revealed both COX-1 and COX-2 to
be expressed in human myometrium (Fig.
4). COX-1 was observed throughout the
tissue and was also localized to single fibroblastic cells embedded in
a field of collagen, as indicated by arrows in Fig. 4A.
COX-1 localization was also apparent in the structural cells
surrounding blood vessels throughout the tissue (data not shown). COX-1
was additionally expressed on smooth muscle cell bundles of the
myometrium, as shown by the arrowhead (Fig. 4A). COX-2 was
highly expressed by individual fibroblasts in the stroma of the
myometrium. Single fibroblasts set in a collagen matrix stained
positive for COX-2 as indicated by arrows (Fig. 4B). This
localization in the tissue correlates with the in vitro data of COX-2
constitutive expression by Thy-1 myometrial fibroblasts.
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PG production by myometrial Thy-1+
and Thy-1 fibroblasts.
PGE2 is a key proinflammatory mediator and is potentially
critical in the reproductive tract for sustaining successful pregnancy and taking part in cervical ripening (17, 45). We
evaluated PGE2 release by myometrial fibroblast subsets
after the cells were left untreated or stimulated for 24 h with
IL-1
. PGE2 production by Thy-1
fibroblasts
was very low (but detectable) in unstimulated cells and, surprisingly,
was not affected by IL-1
treatment. In contrast, basal
PGE2 production by Thy-1+ fibroblasts was high
and was increased twofold by IL-1
treatment (Fig.
5A). Thy-1+ and
Thy-1
fibroblasts were also screened for ability to
produce PGF2
, PGD2, and
PG6-keto-F1
. Whereas Thy-1+ fibroblasts
produced PGF2
and PGD2 in response to
IL-1
, the Thy-1
fibroblasts produced only very low
basal levels of these prostanoids, which were not upregulated by
IL-1
(data not shown).
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ERK1, ERK2, and p38 MAP kinase phosphorylation in myometrial
fibroblast subsets.
Previous studies demonstrated a role for ERK1 (p42) and ERK2 (p44) MAP
kinases in response to IL-1, as well as in the signaling pathway for
COX-2 but not COX-1 (1, 23). The MAP kinase p38 is also
involved in inflammation-type responses and can participate in COX-2
induction (14). We first determined phosphorylation of
ERK1, ERK2, and p38 in the myometrial fibroblast subsets after an
IL-1
time course. The MEK1 inhibitor PD-98059 (50 µM) was included
at each time point to ascertain its blocking effect on phosphorylation
of ERK1/2. Interestingly, the two subsets displayed differential
regulation of ERK1 and ERK2. Thy-1+ fibroblasts showed
sustained phosphorylation of ERK1 and ERK2, which peaked at 5-30
min after IL-1
treatment and subsided by 6 h (Fig.
6A). Thy-1
fibroblasts had a more transient ERK phosphorylation that peaked by 5 min after IL-1
stimulation and disappeared by 15 min (Fig. 6A). Also, in the case of Thy-1
cells, ERK1
was phosphorylated first at the 1 and 2 min time points, whereas ERK2
only peaked at 5 min together with ERK1. PD-98059 inhibited
IL-1
-induced ERK phosphorylation in both subsets. Both myometrial
fibroblast subpopulations showed IL-1
-induced p38 phosphorylation,
even though the p38 time course also varied between the subsets (Fig.
6B). In Thy-1+ fibroblasts, peak p38
phosphorylation was at 15 and 30 min of IL-1
treatment, whereas
Thy-1
fibroblasts showed an earlier and more sustained
p38 phosphorylation, beginning at 1 min of IL-1
stimulation.
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Effect of ERK1, ERK2, and p38 MAP kinase signaling pathways on
COX-1 and COX-2 expression in Thy-1+ and
Thy-1 myometrial fibroblasts.
We next examined whether ERK1/2 or p38 MAP kinases were involved in the
signaling pathway of COX-1 or COX-2 expressed by the myometrial
fibroblast subpopulations. Myometrial fibroblast subsets were treated
with IL-1
(10 ng/ml) for 18 h, with or without the MEK1
inhibitor PD-98059 (50 µM), to determine the effect of ERKs on COX-1
and COX-2 expression. To determine the effect of p38 signaling on COX-1
and COX-2, the downstream p38 inhibitor SB-203580 (50 µM) was
employed. As anticipated, COX-1 expression was not affected by PD-98059
or SB-203580 in either subset (Fig. 7).
IL-1
-induced COX-2 expression was completely abolished by addition
of PD-98059 in Thy-1+ fibroblasts, implicating ERK1 and
ERK2 in its regulation (Fig. 7). However, PD-98059 had no effect on
COX-2 expression in Thy-1
fibroblasts. Therefore, ERKs do
not appear to contribute to constitutive COX-2 expression in this
fibroblast population. SB-203580 also blocked COX-2 expression in
Thy-1+ fibroblasts; thus p38 is also a signaling pathway
utilized for COX-2 induction in this subset. Similar to PD-98059, the
p38 inhibitor SB-203580 had no effect on COX-2 expression in
Thy-1
fibroblasts, also excluding this MAP kinase from
maintaining constitutive COX-2 in Thy-1
cells. The same
results described above on the effect of ERKs and p38 MAP kinase
pathways on COX-1 and COX-2 were observed by using immunocytochemistry
(data not shown).
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DISCUSSION |
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Fibroblasts are dynamic cells whose functions and characteristics
differ according to their anatomic location and the environment to
which they are exposed. Variations in fibroblast responses from tissue
to tissue and within a single tissue can now be explained by the
existence of subsets within a fibroblast population. The data contained
herein support the concept that two types of fibroblasts inhabit the
human myometrium and that these subsets have unique roles in producing
PGs, seminal mediators of inflammatory responses, and reproductive
tract functions. Table 1 summarizes the
characteristics of the Thy-1+ and Thy-1
subsets and their COX and PG profiles. We have established that only
Thy-1+ human myometrial fibroblasts are capable of
producing substantial levels of PGE2, a PG that has
dramatic effects on processes of the reproductive tract.
PGE2 is a vasoactive mediator, participating in leukocyte
recruitment at the site of its production. A role for invading
leukocytes has been postulated in parturition (17). Another important role of PGE2 is its synergy with IL-8 for
neutrophil recruitment (17, 18), which is involved in
cervical ripening, a prerequisite for safe labor (22).
Infiltrating neutrophils are the main source of the collagenase
involved in digesting collagen bundles, leading to the ripening or
softening of the cervix (17). Thy-1+ and
Thy-1
myometrial fibroblasts were recently shown to
produce IL-8 upon stimulation with IL-1
(21).
Thy-1+ myometrial fibroblasts can thus contribute to
processes such as cervical ripening, acting as effectors in the
initiation of the inflammatory response and neutrophil recruitment at
the time preceding parturition.
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We demonstrate that high basal levels of PGE2 in the Thy-1+ subset were mediated through COX-1. This is an extremely interesting finding, because high PGE2 production is usually a consequence of COX-2 upregulation (6). The data reported herein are the first to demonstrate that PGE2 production is mainly mediated by COX-1 in human reproductive fibroblasts. We argue that human myometrial fibroblasts are unique in this respect, further establishing the concept of fibroblast heterogeneity across tissues. Because PGE2 is so critical in the reproductive tract in sustaining pregnancy by maintaining a type 2 cytokine environment, it makes sense that it is mainly synthesized by COX-1, a largely constitutive enzyme. The high COX-1 expression in Thy-1+ fibroblasts allows sustained PGE2 synthesis and designates the Thy-1+ myometrial fibroblast subset as a critical component in maintaining necessary PGE2 homeostasis in the myometrium.
COX-1 and COX-2 expression in Thy-1 myometrial
fibroblasts is also of great interest. The expression pattern of COX-1
and COX-2 in Thy-1
fibroblasts does not follow that
typical of fibroblasts. COX-1 is expressed at barely detectable levels
in unstimulated cells but becomes associated with the nucleus upon
IL-1
treatment. This is a novel and exciting finding, because it is
the first time that we are aware of that COX-1 expression was shown to
localize to the nucleus after stimulation. Its significance, however,
is unclear at present. We speculate that a function of nuclear COX-1 is
the production of PGs that can interact with nuclear receptors, such as
peroxisome proliferator-activated receptors (PPARs). Furthermore, PGE2 released at the nuclear site could interact with
nuclear PGE2 receptors (2, 3) and directly
modulate gene transcription. Further studies are needed to determine
the function of nuclear COX-1-mediated prostanoids and ascertain their
role in the human female reproductive system.
COX-2 expression in Thy-1 myometrial fibroblasts was also
unusual in that it is constitutively expressed in both unstimulated and
IL-1
-treated cells. Recent evidence, however, indicates that some
tissues appear to have constitutive COX-2, such as human thyroid
epithelium (38), rat kidney (13), and stromal
cells in the mouse small intestine lamina propria (30).
Interestingly, constitutive COX-2 expression was also reported in the
rat and human male reproductive tract (20, 27). It appears
that constitutive COX-2 plays a key role in the reproductive system.
Our current findings indicate that COX-2 maintenance in
Thy-1
fibroblasts is ERK1/2 and p38 independent. Thus the
cause of basal COX-2 protein levels in Thy-1
cells
remains to be discovered. A possibility could be an autocrine or
paracrine stimulus that adjusts COX-2 levels in these cells. Future
studies need to assess the importance of constitutive COX-2 in the
human female reproductive tract and determine its regulation in
Thy-1
myometrial fibroblasts.
The lower molecular weight band detected for COX-2 in the
Thy-1 subset (as revealed by Western blot) compared with
the Thy-1+ cells is of interest. Human myometrial tissue
expresses this lower 70-kDa band for COX-2 (data not shown). The lower
band isoform could arise due to a glycosylation change, making it less
capable of metabolizing arachidonic acid. In experiments where
exogenous arachidonic acid was added, the Thy-1
subset
still produced very low levels of PGE2 (data not shown). The Thy-1
myometrial fibroblast subset can, however,
respond to IL-1
by upregulating IL-6 and IL-8 production
(21). PG synthesis also depends on the presence of PG
synthases, whose expression may vary during different events (for
example, pregnancy) in the reproductive tract. Low activity of
PGE2 synthase in Thy-1
fibroblasts could
account for the low PGE2 production. Another possibility is
that this form of COX-2 acts as a preformed "precursor" proenzyme
that awaits a stimulus for its activation and a robust synthesis of PGs.
Labor is associated with increased COX-2 expression in uterine tissues
(29, 43). COX-2 expression is suggested to be responsible for induction of PGF2 (33), which is a
vasoconstrictor and responsible for labor induction in mice
(42). Specifically, the primary role of the myometrium is
to provide contractions during labor. In our studies, the
Thy-1+ myometrial subset was capable of increased
PGF2
production with IL-1
stimulation, and this was
mainly via COX-2, as determined by using COX-selective inhibitors (data
not shown). Given that the COX general inhibitor indomethacin is widely
and successfully used to prevent preterm delivery in humans, a role
implicating COXs is suggested. Furthermore, studies administering the
COX-2 selective drug nimesulide in sheep showed a delay in delivery when labor was induced in ewes (33). COX-2 selective
inhibitors were also shown to inhibit spontaneous uterine contractions
in the rat (34). Our results provide insight to these
reports and suggest the potential use of a COX-2-selective drug to
prevent preterm labor. In this way, fetal well-being is less likely to be compromised, because inhibition of COX-1-derived PGE2
will not occur. Alternatively, premature contractions may be better inhibited by selectively blocking COX-2 and PGF2
.
However, care should be taken in this assessment because the function
of constitutive COX-2 in the myometrium remains to be elucidated.
In conclusion, Thy-1+ and Thy-1 fibroblast
subsets in the female human myometrium are dynamic cell populations
with distinct characteristics of COX subcellular distribution,
regulation, and PG production. The identification of unique fibroblast
subpopulations in the human reproductive tract that have distinct
profiles with respect to inflammatory phenotype underscores the
importance of considering alternate roles of fibroblast subpopulations
in reproductive tract processes. It is likely that each myometrial
fibroblast subpopulation contributes differently to both the
physiological functions and the development of inflammation in the
human reproductive system. Further understanding of fibroblast subset
physiology is imperative, because the overproliferation of one
subpopulation may render a patient more susceptible to a condition
involving an inflammatory process in the human reproductive tract.
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ACKNOWLEDGEMENTS |
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We thank Dr. Kerry O'Banion and Dr. Patricia Sime for critical reading of the manuscript.
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FOOTNOTES |
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This research was supported by United States Public Health Service Grants DE-11390, HL-56002, EY-08976, and ES-01247, the Burroughs-Wellcome Foundation, and the Pepper Center.
Address for reprint requests and other correspondence: R. P. Phipps, Univ. of Rochester School of Medicine and Dentistry, 601 Elmwood Ave, Box 850, MRB-X Rm. 3-11001, Rochester, NY 14642 (E-mail: Richard_Phipps{at}urmc.rochester.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
April 24, 2002;10.1152/ajpcell.00065.2002
Received 14 February 2002; accepted in final form 18 April 2002.
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