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Modulates Prolactin and Tissue Factor Expression in Differentiating Human Endometrial Stromal Cells1
Institute of Reproductive and Developmental Biology, Imperial College School of Medicine, and Medical Research Council Clinical Sciences Center (J.M.), Hammersmith Hospital, London, United Kingdom W12 ONN; and Department of Pathology, Hillingdon Hospital (F.B.), London, United Kingdom UB8 3NN
Address all correspondence and requests for reprints to: Dr. Jan Brosens, Institute of Reproductive and Developmental Biology, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London, United Kingdom W12 ONN. E-mail: j.brosens{at}ic.ac.uk
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Abstract |
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(IFN) released by resident uterine
immune cells are thought to influence the expression of differentiated
function in the human endometrium. Decidualization of the stromal cell
compartment is confined to the superficial endometrial layer in the
nonpregnant uterus. To explore the molecular mechanism underlying the
spatial expression of the decidual phenotype, the effect of IFN on
the induction of two well characterized markers of endometrial stromal
(ES) cell differentiation, PRL and tissue factor (TF), has been
investigated. IFN antagonizes cAMP-mediated PRL protein and
messenger RNA expression in primary ES cell cultures through inhibition
of decidual PRL promoter activity. In parallel, IFN stimulates
Stat-1 (signal transducer and activator of transcription-1) expression,
phosphorylation, and translocation to the nucleus. Exogenously
expressed Stat-1 potently represses decidual PRL promoter activation,
indicating the potential for the inhibitory effects of IFN to be
mediated by Stat-1. We demonstrate that although the coactivator
CREB-binding protein/p300 is essential for decidual PRL transcription,
this coactivator does not appear to be the target for IFN-mediated
repression. By contrast, IFN has little effect on cAMP-mediated TF
expression, but induces TF in ES cells not exposed to a decidualizing
stimulus. This suggested that in vivo TF expression may
not be restricted to decidualizing cells of the superficial layer and
was confirmed by imunohistochemical analysis demonstrating intense TF
staining in the basal stromal compartment during the regeneration phase
of the cycle. The differential sensitivity of
decidualization-associated genes to IFN illustrates its potential
role as a selective biological response modifier that influences
regional function within the endometrium. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role played by estrogen and progestin receptors in mediating the effects of ovarian sex steroids in target organs is now well established. More elusive is a molecular definition of the factors that determine tissue-specific responses to systemic estradiol and progesterone. How, for example, is it possible to achieve within the uterus such spatial diversity to endocrine stimulation? The activation of cell surface receptors and interaction of their signaling molecules with steroid hormone receptors provide one example of how local tissue microenvironments may determine cellular function. Cytokines and growth factors released by endometrial immune cells, including T cells, uterine natural killer (NK) cells, polymorphonuclear neutrophils, macrophages, and monocytes cells, are thought to play a pivotal role in establishing such microenvironments in the human endometrium (2, 3, 4, 5). The spatial and temporal distribution of these cells is also tightly controlled by ovarian hormones. For instance, during the reproductive years lymphoid aggregates, consisting mainly of T cells and a few B cells, are characteristically found in the endometrial-myometrial junction (3, 6, 7). These aggregates are small during the early proliferative phase and significantly increase in size during the second half of the cycle (6, 7). During the proliferative phase the superficial endometrial layer contains only a few uterine NK cells, macrophages, and T cells dispersed throughout the stroma and glands. However, after ovulation the number of uterine NK cells, but not T cells or macrophages, increases dramatically until a few days premenstrually (5).
Interferons (IFNs) are a family of multifunctional cytokines,
originally identified by their ability to confer cellular resistance
against viral infection. Type II interferon, IFN, is an
immunomodulatory T helper 1-type cytokine secreted predominantly by
activated T lymphocytes and NK cells (8). It has been
suggested that IFN in the human endometrium is secreted by lymphoid
aggregates in the basal endometrial layer. Production of IFN is
thought to contribute to the low apoptotic and proliferative activities
in this layer and could account for the higher local expression of
IFN-dependent genes, such as class II major histocompatibility
complex antigens and heat shock protein-70 (2, 3, 4, 9, 10, 11).
We postulated that the absence of a decidualizing response in the basal
stroma could also, at least in part, be mediated by locally expressed
IFN. Decidualization is a differentiation process of the endometrial
stromal compartment essential for blastocyst implantation and placenta
formation. In humans this process is independent of the presence of a
blastocyst and is first apparent in the second half of the luteal phase
in stromal cells around the spiral arterioles and capillaries of the
superficial layer. At the end of the menstrual cycle the upper part of
the superficial layer is crowded with decidualized stromal cells.
Several studies have suggested that endometrial stromal (ES) cell
differentiation in response to estradiol and progesterone results from
sustained elevation of intracellular cAMP levels after the release of
local factors such as relaxin, PGE2, and CRF
(12, 13, 14, 15).
The objective of this study was to determine whether IFN is capable
of modulating biochemical features of decidual differentiation. Primary
culture of human ES cells induced to express PRL and tissue factor
(TF), two well established end points of decidualization
(12, 13, 14, 15, 16, 17), were therefore investigated for the inhibitory
activity of IFN. We provide evidence that IFN is a potent
inhibitor of decidual PRL expression, but these effects do not extent
to TF. The divergent spatial and temporal expression of these two genes
in endometrial tissues may therefore be related to their sensitivity to
modulatory cytokine influences that underlie regional differentiation
of the endometrium.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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was purchased from
R & D Systems (Oxon, UK).
Primary ES cell culture
ES cells were isolated from normal proliferative endometrial
tissues obtained from cycling women by endometrial biopsy at the time
of diagnostic laparoscopy and hysteroscopy. Hammersmith and Queen
Charlotte’s Hospital research and ethics committee approved the study,
and patient consent was obtained before biopsy. Samples were collected
in Earle’s buffered saline containing 100 U/ml penicillin and 100
µg/ml streptomycin. The tissues were washed twice in DMEM/F12, finely
minced, and enzymatically digested with collagenase (134 U/ml) and
deoxyribonuclease type I (156 U/ml) for 1 h at 37 C. After
centrifugation at 400 x g for 4 min, the pellet was
resuspended in maintenance medium of DMEM/F12, 10% (wt/vol)
dextran-coated charcoal-treated FBS (DCC-FBS), 1% (wt/vol)
L-glutamine, and 1% (vol/vol) antibiotic-
antimycotic solution. ES cells were separated from epithelial cells
and passed into culture as described previously (12, 13).
Proliferating ES cells were cultured in maintenance medium until
confluence. Confluent monolayers were treated in DMEM/F12 containing
2% (vol/vol) DCC-FBS with 0.5 mM 8-bromo-cAMP
and/or 10-6 M MPA. All experiments were carried out before the fourth cell passage.
PRL and DNA assays
PRL levels in supernatants were measured by microparticle enzyme
immunoassay (MEIA, AxSYM system, Abbott Laboratories,
Chicago, IL). The coefficient of variation within assays was 2–3%,
and that between assays was 6–8%. DMEM/F12 supplemented with DCC-FBS
did not show measurable PRL concentrations. PRL levels were normalized
to the DNA content of each culture flask at the end of the treatment
period. The DNA content was measured by quantitative fluorometric
analysis at room temperature. Cells were solubilized with 0.02%
(wt/vol) SDS. Aliquots were then mixed with 1 µg/ml Hoechst 33258 in
SSC (1 x standard saline citrate), and fluorescence was measured
in a fluorometer at 344 nm excitation and 460 nm. Calf thymus DNA was
used as standard.
SDS-PAGE, Western blotting, and immunodetection
A modified method of Rittenhouse and Marcus (18)
was used for protein analysis. Protein concentrations were determined
by Bradford assay (Bio-Rad Laboratories, Inc., Hemel
Hempstead, UK). Equal amounts of nuclear and cytosolic proteins (20
µg) were separated on a 7.5% SDS-polyacrylamide gel before
electrotransfer at 80 V onto a polyvinylidene difluoride membrane
(Hybond-P, Amersham Pharmacia Biotech). Even loading and
transfer efficiency were confirmed by Ponceau S staining. Nonspecific
binding sites were blocked with 0.2% (wt/vol) I-Block (Tropix,
Bedford, MA) in PBS with 0.1% (vol/vol) Tween at room temperature for
2 h. The primary antibody, rabbit polyclonal anti-TF antibody, was
a gift from Dr. J. McVeigh. Rabbit polyclonal anti-Stat-1 and rabbit
polyclonal phospho (Ser727)Stat-1 were purchased
from Upstate Biotechnology, Inc. (Lake Placid, NY). The
secondary antibody, peroxidase-conjugated goat antirabbit IgG, was
obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA). Blots were exposed to primary antibodies diluted in PBS-Tween for
1 h at 4 C and then incubated with secondary peroxidase-conjugated
antibody for 1 h at 4 C. Protein bands were visualized by enhanced
chemiluminescence (ECL Western Blotting Detection, Amersham Pharmacia Biotech).
RT-PCR and Southern blotting analysis
Total RNA was extracted from ES cells with RNAzol B
(Biogenesis). One microgram of total RNA was reverse
transcribed and amplified in a single reaction using Access RT-PCR
System (Promega Corp.) according to the manufacturer’s
instructions. Simultaneous amplification of decidual PRL and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed by
adding 10 pmol of each of the following oligonucleotides to each
reaction: decidual PRL sense (5'-CCTTCGAGACCTGTTTGACC-3'), decidual PRL
antisense (5'-AAAACTTAGGTACGAACACC-3'), GAPDH sense
(5'-CCACCCATGGCAAATTCCAT-3'), and GAPDH antisense
(5'-AGTGGGGACACGGAAGGCCA-3'). The GAPDH complementary DNA (cDNA),
representing a nonregulated gene, served as an internal control. The
reaction was allowed to continue for 28 cycles, which was within the
exponential phase of the amplification reaction, as determined by cycle
profiling. Southern blots of the PCR products were successfully
hybridized with an internal 32P-labeled
oligonucleotide complementary to the decidual PRL PCR product
(5'-GGAGCTGATAGTCAGCCAGG-3'), followed by a
32P-labeled GAPDH sense oligonucleotide.
Reporter constructs, expression vectors, and transient transfection
studies
The reporter vectors decidual PRL-3000/luc and decidual
PRL-332/luc, carrying 3000 and 332 bp, respectively, of 5'-flanking DNA
to the decidual-specific promoter of the hPRL gene, were provided by
Dr. Birgit Gellersen (Hamburg, Germany). Expression vectors for
CREB-binding protein (CBP) and 12S-E1A were obtained from Dr. Malcolm
Parker (London, UK). Plasmid pRSV-C
, encoding the protein kinase A
(PKA) -catalytic subunit, was a gift from Dr. Richard Maurer
(Portland, OR). The expression vector for Stat-1 (p91; signal
transducer and activator of transcription-1) was a gift from Dr.
Bernd Groner (Frankfurt, Germany). The plasmid encoding p300 was
obtained from Dr. Nick Jones (London, UK).
Transient transfections of ES cells plated at a density of 5 x 105 cells/well in 12-well plates were performed by the calcium phosphate precipitation in medium supplemented with 2% DCC-FBS. Details of the transfection protocol and the treatments are indicated in the figure legend. Cell extracts were harvested, and luciferase activity was measured with the luciferase reagent kit (Promega Corp.) and expressed as relative light units. Transfections were performed in triplicate and repeated at least three times. Representative experiments are shown (mean ± SD).
Immunohistochemistry
Archival paraffin-embedded, formalin-fixed, full-thickness
endometrial specimens were examined for TF immunoreactivity. A total of
12 uteri were examined. All specimens were from cycling, premenopausal
women and were free of uterine disease, such as adenomyosis. Using
standard criteria (19), endometria were allocated to the
menstrual phase (n = 3), the proliferative phase (n = 3), the
early secretory phase (n = 2), the midsecretory phase (n =
1), or the late secretory phase (n = 3). Five-micron sections
placed on 1% (wt/vol) polysine slides (Merck Ltd., Poole, UK) were
deparaffinized, dehydrated, exposed to 0.3% (vol/vol)
H2O2 for 15 min, and
subsequently microwaved in 0.01 M citrate buffer, pH 6.0.
Immunostaining was carried using antihuman tissue factor rabbit Ig
polyclonal antibody, biotinylated swine antirabbit Ig (DAKO Corp., High Wycombe, UK; diluted 1:500), and peroxidase-labeled
streptavidin (Roche Molecular Biochemicals, Lewes, UK;
diluted 1:500). Controls were performed by replacing the primary
antibody with rabbit nonimmune serum.
Statistical analysis
All data are expressed as the mean ± SD and
were analyzed for significance using Student’s t test or
ANOVA as appropriate. P 
0.05 was the minimum
criteria for declaring significance.
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Results |
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inhibits decidual PRL gene expression in differentiating ES
cells markedly inhibited cAMP- induced PRL protein expression
in differentiating ES cells in a dose-dependent manner and completely
abolished the synergistic effect of MPA (Fig. 1A
). These inhibitory effects were
apparent at very low IFN concentrations (0.1–0.01 ng/ml),
emphasizing the potency of this cytokine as a negative regulator of
decidual PRL expression.
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on PRL protein expression was
accompanied by a dose-dependent repression of the cAMP-induced increase
in decidual PRL messenger RNA, measured by semiquantitative RT-PCR
(Fig. 1B
). To confirm that IFN antagonism of PRL protein and
messenger RNA expression reflected inhibition of transcription, the
effect of IFN on the decidual PRL promoter (dPRL-3000/luc)
activation was investigated by transient transfection. Promoter
activity induced by cAMP was inhibited by IFN in a dose-dependent
manner (Fig. 1C), confirming that IFN is a potent negative regulator
of cAMP-induced decidual PRL gene transcription in human ES cells. The
minimal flanking region that retains sensitivity to cAMP (dPRL-332/luc)
(14, 20) also retained sensitivity to inhibition by IFN
(Fig. 1D).
Activation of Stat-1 in differentiating ES cells in response to
IFN
Binding of IFN to its cognate cell surface receptor is thought
to activate, through targeted tyrosine phosphorylation, Stat-1, the
founding member of the STAT family of transcription factors
(21, 22, 23). Stat-1 activation requires phosphorylation of a
single residue, Tyr701, that is essential for its
dimerization, nuclear translocation, and binding to specific DNA
elements in the promoter region of target genes.
Before stimulation with cAMP, undifferentiated ES cells expressed low
levels of Stat-1, which was predominantly cytoplasmic; Stat-1 abundance
in the nucleus was considerably lower, and only a minor component of
this was phosphorylated (Fig. 2). Upon
treatment with cAMP, the abundance of Stat-1, cytoplasmic and nuclear,
was significantly decreased, and the phosphorylated form of nuclear
Stat-1 was no longer apparent (Fig. 2). The cAMP effect on cellular
Stat-1 was reversed by the addition of MPA. This may reflect enhanced
expression of other known activators of the Stat-1 signaling pathway,
such as PRL, epidermal growth factor, and their respective receptors,
in cultures treated with cAMP plus MPA (24, 25, 26).
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increased cytoplasmic and nuclear Stat-1
abundance together with an increase in the prominence of nuclear
phospho-Stat-1. This increase in abundance of Stat-1 and of the nuclear
phosphorylated form in response to IFN was equally apparent in cells
that had or had not been cotreated with cAMP (Fig. 2).
Stat-1 inhibits decidual PRL promoter activity
To provide further evidence for the role of Stat-1 in mediating
IFN repression of the decidual PRL promoter, ES cells were
transiently transfected with dPRL-3000/luc reporter construct, and
expression vectors for the catalytic -subunit (pC) of the PKA
holoenzyme, Stat-1, or both. Previous studies have shown that the
catalytic subunit of the PKA holoenzyme mediates the cAMP response in
ES cells (13, 14). Figure 3
demonstrates that coexpression of Stat-1 markedly inhibited
PKA-dependent decidual PRL promoter activity and that this inhibition
was further enhanced by the addition of IFN, suggesting that
activation of IFN receptors and recruitment of signaling
intermediates supplement the activity of exogenously expressed
Stat-1.
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activation sequence (GAS) in the promoter region of target genes, and
thereby regulate the transcription of these genes
(21, 22, 23). However, analysis of the minimal cAMP-responsive
decidual PRL promoter region (-332/+11) using the TFSEARCH program did
not show the presence of GAS elements. Hence, we postulated that Stat-1
could exert its inhibitory action on the decidual PRL promoter through
interaction with other essential DNA-binding proteins or
non-DNA-binding proteins, such as coactivators. The most obvious
candidate coactivator relevant to the decidual PRL promoter was
CBP/p300, as it is capable of binding and integrating nuclear hormone
receptors and diverse classes of exogenously regulated transcription
factors including Stat-1 (27, 28, 29). The importance of
CBP/p300 in the regulation of the decidual PRL promoter was initially
demonstrated by the ability of the adenovirus 12S-E1A protein (Fig. 4A), a factor that inactivates the
coactivator function of the endogenous CBP/p300, to inhibit
cAMP-dependent activation (30, 31). The synergy between
cAMP and MPA on the promoter activity was also lost, indicating that
CBP/p300 is indeed essential for decidual PRL gene transcription.
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-regulated Stat-1, the inhibitory effect of IFN could not be
relieved by overexpression of p300 (Fig. 4B). Paradoxically, p300
inhibited cAMP-dependent activation of the decidual PRL promoter in a
dose-dependent manner. This may reflect squelching of limiting
amounts of a factor(s) obligatory to decidual PRL gene expression. In
contrast, p300 enhanced ligand-bound progesterone receptor B
trans-activation of the mouse mammary tumor virus promoter
(MMTV-luc), a classical progesterone receptor-responsive promoter,
indicating that its inhibitory effect on the dPRL-3000/luc is promoter
specific (data not shown).
IFN induces tissue factor (TF) expression in ES cells
We next determined whether the inhibitory effects of IFN were
limited to the expression of PRL or also affected other biochemical
markers of decidualization. TF is a 262-amino acid membrane-bound
glycopeptide whose expression in ES cells, in vivo and
in vitro, mimics that of decidual PRL (13, 16, 17). Serum factors are known to induce TF expression in several
cell types, including ES cells, and consequently confluent cultures
were maintained in low serum (2% DCC-FBS) for 48 h before
treatment with cAMP, IFN, or their combination. Sustained activation
of the PKA pathway induced TF expression in differentiating ES cells,
but cotreatment with IFN had little effect on TF expression (Fig. 5). Also in contrast to its effect on PRL
secretion, IFN induced TF expression in undifferentiated cells (Fig. 5).
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, in vitro would, if the hypothesis of cytokine
gradients within the endometrium is correct, predict that its
expression is not confined to predecidualized ES cells in the
superficial layer that selectively express PRL. Full-thickness
endometria obtained at different phases of the cycle were
immunohistochemically stained for TF. As reported previously, during
the late secretory phase TF expression in the stromal compartment was
predominantly confined to the predecidualized cells in the upper part
of superficial layer (Fig. 6A). However,
moderate staining was also apparent in the basal stromal compartment
near the endometrial-myometrial junction (Fig. 6B). Intriguingly,
endometrial epithelial cells also express TF, but its expression was
almost exclusively confined to the luminal epithelial cells. Often, a
sharp demarcation was noted between TF-positive luminal and TF-negative
glandular epithelial cells near the introitus of the glands (Fig. 6C).
TF expression in the stromal compartment was weak during the
proliferative and early secretory phases of the cycle in both
endometrial layers (data not shown). However, the most intense
expression of TF was seen in the basal endometrial layer during the
late menstrual or regeneration phase of the cycle (Fig. 6D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-signaling pathway antagonizes
PKA-dependent decidual PRL gene expression in differentiating ES cells
through inhibition of transcription. This response to IFN is
dependent upon induction, tyrosine phosphorylation, dimerization, and
nuclear translocation of Stat-1. Promoter analysis showed that the
minimal cAMP-responsive decidual PRL promoter (dPRL-332) confers the
IFN-mediated repression, although this region is devoid of known
high affinity binding sites for activated Stat-1. It is, however, known
that some of the antiviral and growth inhibitory properties of IFN
do not require binding of Stat-1 to cis elements in the
promoter region of target genes, but result from competition between
Stat-1 and other classes of transcription factors for limited amounts
of essential coactivators (32, 33). For instance, Horvai
and co-workers demonstrated that activated Stat-1 represses
transcription of the macrophage scavenger receptor gene by inhibiting
recruitment of CBP/p300-containing coactivator complexes
(33). CBP/p300 has potent histone acetyltransferase
activity that is essential for inducing a more transcriptionally active
chromatin conformation. In addition, CBP/p300 promotes interactions
between the RNA polymerase II basal transcription complex and diverse
classes of sequence-specific transcriptional activators (28, 29). We postulated that the presence of CBP/p300 on the decidual
PRL promoter could serve as a point of convergence for the various
factors essential for the coordinated expression of PRL. To test this
hypothesis, primary ES cell cultures were transiently transfected with
the adenovirus E1A oncoprotein, which binds and inactivates the
coactivator function of CBP/p300 (30, 31). In
differentiating ES cells, transient expression of E1A abrogated
cAMP-mediated decidual PRL promoter activity. In untreated ES cells,
the basal reporter gene transcription was not affected by E1A,
suggesting that CBP/p300 is actively recruited to the decidual PRL
promoter upon PKA activation. Paradoxically, overexpression of CBP/p300
inhibited cAMP-mediated activation of the decidual PRL promoter. This
repression was promoter specific and may reflect the ability of
exogenously expressed CBP/p300 to squelch obligatory endogenous factors
and thereby impair the formation of a transcriptionally active complex.
Steroid receptor coactivator-1, which is essential for the decidual
response both in vivo and in vitro (13, 34), is potentially such a factor. Furthermore, CBP/p300 failed
to counteract IFN-mediated inhibition of decidual PRL promoter
activity in response to cAMP. This suggests that repression of the cAMP
response by Stat-1 does not involve sequestration of CBP/p300.
We are currently investigating several other potential mechanisms of
suppression. For instance, although the proximal decidual PRL promoter
does not contain IFN-responsive GAS motifs, this does not eliminate
the possibility that Stat-1 competes with other transcription factors
for site occupancy and thereby interferes with the formation of a
transcriptionally active multimeric complex. Second, it is also
conceivable that Stat-1-dependent suppression requires the induction of
a new gene product with repressor activity.
Third, studies have shown that IFN can activate signaling pathways
other than Stat-1, such as the MAP kinase pathway (35).
Hence, it appears possible that additional factors are involved in
Stat-1 transrepression of the decidual PRL promoter. A final line of
inquiry is based on the observation that induction, activation, and
nuclear translocation of other STAT family members, Stat-5a and
Stat-5b, are essential for sustaining and enhancing the decidual
phenotype in human ES cells (our manuscript in preparation).
This observation raises the possibility that Stat-5 and Stat-1 could
compete for a cytoplasmic factor capable of modulating STAT-dependent
gene transcription in the nucleus. The recently described N-Myc
interacting protein, Nmi, is potentially such a factor
(36). Although it lacks an intrinsic
trans-activation domain, Nmi has been shown to interact with
Stat-1 and Stat-5, to stabilize Stat-CBP complexes, and to enhance
Stat-dependent gene transcription. Nmi is expressed in a variety of
tissues, including the uterus (37).
This study unequivocally demonstrates that IFN antagonizes decidual
PRL expression in differentiating primary ES cell cultures. This does
not necessary imply an a priori role for this cytokine in
abrogating the decidual response in the basal stromal compartment.
First, the human endometrium is known to express several other factors
capable of inhibiting decidual PRL expression in vitro,
including lipocortin-1, retinoic acid, transforming growth factor-ß1,
endothelins, and certain cytokines produced by decidual immune cells
such as interleukin-1 and tumor necrosis factor-
(38, 39, 40, 41, 42, 43). Second, only a few studies have examined IFN
protein expression in the nonpregnant human uterus, and although there
is agreement that IFN immunoreactivity does not vary with the stage
of the cycle, no consensus exists on its spatial expression or the
identity of IFN-producing cell types in the endometrium. Stewart
et al. (3) reported that the lymphoid
aggregates in the basal endometrial layer are the major source of
immunoreactive IFN in the human endometrium. Yeaman et
al. (6), using a culture system of fresh uterine
sections, found no IFN staining in lymphoid aggregates in the
absence of exogenous stimuli and identified polymorphonuclear
neutrophils, located below the luminal epithelium and adjacent to the
glandular epithelium, as the source of constitutive stromal IFN
production. Although the reasons for these discrepancies are unclear,
it should be noted that the many of the uterine specimens examined in
the latter study contained adenomyosis, a disease characterized by loss
of normal uterine polarity and aberrant expression of T helper 1 cell
cytokine-inducible genes (44, 45). Finally, in our culture
system IFN failed to antagonize the gross morphological
transformation of spindle-shaped undifferentiated ES cells into rounded
decidual cells (data not shown). Furthermore, IFN did also not
repress TF expression in response to cAMP treatment. Together these
observations indicate that endometrial IFN is more likely to play a
role in modulating, rather than repressing, ES cell
differentiation.
TF is a cell membrane-bound glycoprotein that initiates hemostasis by
complexing with the zymogen serine protease factor VII (FVII) to
activate the intrinsic and extrinsic coagulation factors, IX and X,
respectively (46). In human endometrium, TF expression has
been reported to be closely associated with decidual transformation and
is often used as a biochemical marker of this differentiation process
(13, 16, 17). The distinct temporal and spatial expression
of TF in the endometrium indicates a pivotal role in maintaining
vascular integrity before menstruation and, if pregnancy occurs, during
intravascular trophoblast invasion (16, 17). We previously
reported that TF expression in primary cultures mimics that of PRL,
characterized by rapid induction in response to cAMP treatment, which
is further enhanced by the addition of progestins. IFN has little
effect on TF expression associated with decidual transformation, but
induces this membrane-bound protein in undifferentiated cells. In
endometrium TF expression is thought to be regulated by the
transcription factor Sp-1 (47), and it is noteworthy that
with regard to the positive effects of IFN, Stat-1 cooperates with
Sp-1 in the regulation of certain target promoters (23).
Furthermore, IFN and other proinflammatory cytokines are potent
positive regulators of TF expression in other cell systems such as
monocytes (48, 49).
The induction of TF by IFN in undifferentiated ES cells suggested
that endometrial TF expression may not be restricted to decidualizing
cells in the superficial layer. Immunohistochemical studies confirmed
that TF is also highly expressed in the basal layer during the
menstrual phase of the cycle. Recent evidence suggests that TF bound to
its ligand may have functions beyond controlling fibrin-dependent
hemostasis and can activate intracellular signaling pathways via the
short cytosolic domain of TF (50). Using cDNA arrays,
Camerer and co-workers (51) demonstrated that interaction
of activated FVII with TF results in up-regulation of genes involved in
a wound-type response. Hence, TF expression during the menstrual phase
may play an active role not only in hemostasis, but also in cyclic
generation of the endometrium. Intense TF immunoreactivity was also
apparent in the surface epithelium throughout the cycle. In contrast,
TF was not detectable in the majority of glandular epithelial cells.
The significance of this finding is at present unclear. However,
activated FVII has been shown to be present in seminal plasma
(52), which opens the exciting possibility that semen
transported through the reproductive tract could alter gene expression
in the adjacent endometrial surface epithelium.
In conclusion, we have demonstrated that the nature of the endometrial
response to IFN is gene specific. The potent inhibition of PRL
expression provides a paradigm that illustrates the capacity of IFN
to modulate ES cell decidualization. In addition, IFN induces TF
expression in undifferentiated ES cells. The distinct temporal and
spatial expression of TF in the human endometrium suggests its role not
only in local hemostasis, but also in tissue regeneration, which is
essential for maintenance of endometrial function.
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Acknowledgments |
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Footnotes |
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Received December 7, 2000.
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References |
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prolactin. Endocrinology 134:353–357This article has been cited by other articles:
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 T. Strowitzki, A. Germeyer, R. Popovici, and M. von Wolff The human endometrium as a fertility-determining factor Hum. Reprod. Update, September 1, 2006; 12(5): 617 - 630. [Abstract] [Full Text] [PDF]
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S. Labied, T. Kajihara, P. A. Madureira, L. Fusi, M. C. Jones, J. M. Higham, R. Varshochi, J. M. Francis, G. Zoumpoulidou, A. Essafi, et al. Progestins Regulate the Expression and Activity of the Forkhead Transcription Factor FOXO1 in Differentiating Human Endometrium Mol. Endocrinol., January 1, 2006; 20(1): 35 - 44. [Abstract] [Full Text] [PDF]
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 A. Katayama, T. Ogino, N. Bandoh, S. Nonaka, and Y. Harabuchi Expression of CXCR4 and Its Down-Regulation by IFN-{gamma} in Head and Neck Squamous Cell Carcinoma Clin. Cancer Res., April 15, 2005; 11(8): 2937 - 2946. [Abstract] [Full Text] [PDF]
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 S. Kato, M. Pinto, A. Carvajal, N. Espinoza, C. Monso, A. Sadarangani, M. Villalon, J. J. Brosens, J. O. White, J. K. Richer, et al. Progesterone Increases Tissue Factor Gene Expression, Procoagulant Activity, and Invasion in the Breast Cancer Cell Line ZR-75-1 J. Clin. Endocrinol. Metab., February 1, 2005; 90(2): 1181 - 1188. [Abstract] [Full Text] [PDF]
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 V Rider, T Potapova, G Dai, and M J Soares Stimulation of a rat uterine stromal cell line in culture reveals a molecular switch for endocrine-dependent differentiation J. Endocrinol., January 1, 2005; 184(1): 119 - 127. [Abstract] [Full Text] [PDF]
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G. Zoumpoulidou, M. C. Jones, S. F. de Mattos, J. M. Francis, L. Fusi, Y. S. Lee, M. Christian, R. Varshochi, E. W.-F. Lam, and J. J. Brosens Convergence of Interferon-{gamma} and Progesterone Signaling Pathways in Human Endometrium: Role of PIASy (Protein Inhibitor of Activated Signal Transducer and Activator of Transcription-y) Mol. Endocrinol., August 1, 2004; 18(8): 1988 - 1999. [Abstract] [Full Text] [PDF]
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 E. Garzia, S. Borgato, V. Cozzi, P. Doi, G. Bulfamante, L. Persani, and I. Cetin Lack of expression of endometrial prolactin in early implantation failure: a pilot study Hum. Reprod., August 1, 2004; 19(8): 1911 - 1916. [Abstract] [Full Text] [PDF]
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I. Y. H. Mak, J. J. Brosens, M. Christian, F. A. Hills, L. Chamley, L. Regan, and J. O. White Regulated Expression of Signal Transducer and Activator of Transcription, Stat5, and its Enhancement of PRL Expression in Human Endometrial Stromal Cells in Vitro J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2581 - 2588. [Abstract] [Full Text] [PDF]
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P = 0.001–0.01 compared with cAMP- plus
MPA-treated cells in the absence of IFN
