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Endocrinology Vol. 142, No. 7 3142-3151
Copyright © 2001 by The Endocrine Society

ARTICLES

Interferon-{gamma} Modulates Prolactin and Tissue Factor Expression in Differentiating Human Endometrial Stromal Cells1

Mark Christian, Petros Marangos, Ian Mak, John McVey, Fred Barker, John White and Jan J. Brosens

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cytokines such as interferon-{gamma} (IFN{gamma}) 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{gamma} on the induction of two well characterized markers of endometrial stromal (ES) cell differentiation, PRL and tissue factor (TF), has been investigated. IFN{gamma} antagonizes cAMP-mediated PRL protein and messenger RNA expression in primary ES cell cultures through inhibition of decidual PRL promoter activity. In parallel, IFN{gamma} 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{gamma} 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{gamma}-mediated repression. By contrast, IFN{gamma} 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{gamma} illustrates its potential role as a selective biological response modifier that influences regional function within the endometrium.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE UTERUS IS composed of heterogeneous cell types that undergo cyclic synchronized waves of proliferation and differentiation in response to the rise and fall of ovarian estrogen and progesterone. In addition to these temporal changes, the human endometrium is further characterized by profound spatial differences in its response to ovarian hormones. During the follicular phase of the cycle, the glandular, stromal, and vascular endothelial cells show a prominent proliferative response in the superficial, but not the basal, endometrial layer (1, 2). After ovulation, the superficial layer undergoes extensive tissue remodeling in response to ovarian progesterone levels, a process that initially involves secretory transformation of the glandular epithelium, followed by edema and decidualization of the stromal compartment. These classical sex steroid hormone responses are absent in the basal endometrial layer, which is characterized by low proliferative activity, absence of glandular secretory transformation, and lack of a predecidualization reaction in the late luteal phase of the cycle.

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{gamma}, is an immunomodulatory T helper 1-type cytokine secreted predominantly by activated T lymphocytes and NK cells (8). It has been suggested that IFN{gamma} in the human endometrium is secreted by lymphoid aggregates in the basal endometrial layer. Production of IFN{gamma} is thought to contribute to the low apoptotic and proliferative activities in this layer and could account for the higher local expression of IFN{gamma}-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{gamma}. 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{gamma} 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{gamma}. We provide evidence that IFN{gamma} 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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Phenol red-free DMEM-Ham’s F-12 mixture (DMEM/F12), collagenase (type I), deoxyribonuclease (type I), 8-bromo-cAMP, medroxyprogesterone acetate (MPA), Hoechst dye 33258 (bisenzimidine), calf thymus DNA, and antibiotic-antimycotic solution were all obtained from Sigma (Poole, UK). FBS and L-glutamine were purchased from Life Technologies, Inc. (Uxbridge, UK). The RNA extraction reagent RNAzol B was obtained from Biogenesis (Bournemouth, UK). Access RT-PCR System, ProFection Mammalian Transaction, and Luciferase Assay System were purchased from Promega Corp. (Southampton, UK). Plasmid Maxi Kits were obtained from QIAGEN (Crawley, UK). Hybond P and Hybond N membranes, Ready-To-Go T4 polynucleotide kinase, and ECL Western blotting detection reagents were obtained from Amersham Pharmacia Biotech (St. Albans, UK). IFN{gamma} 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{alpha}, encoding the protein kinase A (PKA) {alpha}-catalytic subunit, was a gift from Dr. Richard Maurer (Portland, OR). The expression vector for Stat-1{alpha} (p91; signal transducer and activator of transcription-1{alpha}) 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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IFN{gamma} inhibits decidual PRL gene expression in differentiating ES cells
We previously reported that human ES cells secrete PRL in vitro in response to treatment with a stable cAMP analog, 8-bromo-cAMP (12, 13). Cotreatment with the progestin MPA further enhances decidual PRL gene expression. Here, we demonstrate that IFN{gamma} 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. 1AGo). These inhibitory effects were apparent at very low IFN{gamma} 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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  		Figure 1. IFN{gamma} inhibits decidual PRL expression. A, Confluent primary ES cells were treated for 96 h in phenol-red free medium containing 2% DCC-FBS with 0.5 mM 8-bromo-cAMP, 8-bromo-cAMP plus IFN{gamma} (concentrations ranging from 0.01–10 ng/ml), or 0.5 mM 8-bromo-cAMP plus 1 µM MPA in the presence or absence of IFN{gamma} (concentrations ranging from 0.01–10 ng/ml). The medium was changed after 48 h. The results show the mean PRL secretion (±SD) of triplicate determinations, corrected for the DNA content in each well. *, P = 0.001–0.05 compared with cAMP-treated cells in the absence of IFN{gamma}. {dagger}, P = 0.001–0.01 compared with cAMP- plus MPA-treated cells in the absence of IFN{gamma}. B, Confluent primary ES cells were treated for 96 h in 2% DCC-FBS with 0.5 mM 8-bromo-cAMP in the presence and absence of IFN{gamma} (0.01–10 ng/ml). Total RNA was extracted and used in simultaneous RT-PCR amplifications, performed in a single reaction for PRL and GAPDH cDNAs. The Southern blot of PCR products was successively hybridized with oligonucleotide probes specific for PRL and GAPDH. C, cAMP-primed ES cells (48 h) were transiently transfected with dPRL-3000/luc (1 µg/well). Subsequently, the cultures were treated with 0.5 mM 8-bromo-cAMP or 0.5 mM 8-bromo-cAMP plus IFN{gamma} at various concentrations. Luciferase activity was measured after 40 h of treatment, and the results show the mean activity (±SD) of triplicate measurements. *, P = 0.001–0.01 compared with cAMP-treated cells in the absence of IFN{gamma}. D, ES cells were transfected with dPRL-332/luc (1 µg/well) and pRSV-C{alpha}, encoding the catalytic subunit of PKA. Luciferase activity was measured after 40 h of treatment in the absence and presence of 10 ng/ml IFN{gamma}, and the results show the mean activity (±SD) of triplicate measurements. *, P = 0.001–0.01 compared with cells transfected with pRSV-C{alpha} in the absence of IFN{gamma}.

 
The inhibitory effect of IFN{gamma} 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. 1BGo). To confirm that IFN{gamma} antagonism of PRL protein and messenger RNA expression reflected inhibition of transcription, the effect of IFN{gamma} on the decidual PRL promoter (dPRL-3000/luc) activation was investigated by transient transfection. Promoter activity induced by cAMP was inhibited by IFN{gamma} in a dose-dependent manner (Fig. 1CGo), confirming that IFN{gamma} 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{gamma} (Fig. 1DGo).

Activation of Stat-1 in differentiating ES cells in response to IFN{gamma}
Binding of IFN{gamma} 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. 2Go). 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. 2Go). 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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  		Figure 2. Induction and subcellular localization of Stat-1 in differentiating ES. Primary ES cells cultured in 2% DCC-FBS were untreated (control) or treated with 8-bromo-cAMP (0.5 mM), MPA (1 µM), IFN{gamma} (1 ng/ml), or a combination of these treatments. After 48 h, protein was extracted from the cytosolic and nuclear compartments, resolved by SDS-PAGE (15 µg protein loaded), and blotted onto polyvinylidene difluoride membrane. Stat-1 protein expression in each cellular compartment was detected using rabbit polyclonal anti-Stat-1 IgG, and phospho-Stat-1 was detected with rabbit polyclonal anti-phospho-(Ser727)-Stat-1 antibody, at a concentration of 1 µg/ml. Bands were visualized using the secondary antibody, peroxidase-conjugated antirabbit IgG (developed in goat), at a concentration of 1 µg/ml, followed by enhanced chemiluminescence.

 
Treatment with IFN{gamma} 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{gamma} was equally apparent in cells that had or had not been cotreated with cAMP (Fig. 2Go).

Stat-1 inhibits decidual PRL promoter activity
To provide further evidence for the role of Stat-1 in mediating IFN{gamma} repression of the decidual PRL promoter, ES cells were transiently transfected with dPRL-3000/luc reporter construct, and expression vectors for the catalytic {alpha}-subunit (pC{alpha}) 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 3Go 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{gamma}, suggesting that activation of IFN{gamma} receptors and recruitment of signaling intermediates supplement the activity of exogenously expressed Stat-1.



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  		Figure 3. IFN{gamma} and Stat-1 inhibit PKA-stimulated transcription of dPRL. A, cAMP-primed ES cells (48 h) were transiently transfected with dPRL-3000/luc (1 µg/well) and pRSV-C{alpha} (0.4 µg/well) encoding the catalytic subunit of PKA and/or with Stat-1 (0.4 µg/well) or the empty vector pSG5 (0.4 µg/well). Cells were untreated (control) or treated with 1 ng/ml IFN{gamma}. Luciferase activity was measured after 40 h of treatment, and the results show the mean activity (±SD) of triplicate measurements. *, P = 0.001–0.01 compared with cells transfected with pRSV-C{alpha} in the absence of Stat-1 and treated with IFN{gamma}.

 
Role of CBP/p300 in Stat-1-mediated repression of the decidual PRL promoter
Activated STATs translocate into the nucleus, bind IFN{gamma} 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. 4AGo), 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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  		Figure 4. Role of CBP/p300 in Stat-1-mediated repression of the dPRL promoter. A, cAMP-primed ES cells (48 h) were transfected with dPRL-3000/luc (1 µg/well) and pJA3 (0.4 µg/well), an expression vector encoding for 12S-E1A, or with the empty vector pSG5 (0.4 µg/well). E1A binds and inactivates CBP/p300. The transfected cultures received no further treatment (control) or were treated with 0.5 mM 8-bromo-cAMP, 10-6 M MPA, or their combination. B, cAMP-primed ES cells (48 h) were transfected with dPRL-3000/luc (1 µg/well), and increasing concentrations of p300 as indicated. Luciferase activity was measured after 40 h of treatment, and the results indicate the mean activity (±SD) of triplicate measurements.

 
Despite this demonstration of the importance of CBP/p300 to activation of dPRL-3000/luc, and hence its potential as a target for inhibition by IFN{gamma}-regulated Stat-1, the inhibitory effect of IFN{gamma} could not be relieved by overexpression of p300 (Fig. 4BGo). 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{gamma} induces tissue factor (TF) expression in ES cells
We next determined whether the inhibitory effects of IFN{gamma} 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{gamma}, or their combination. Sustained activation of the PKA pathway induced TF expression in differentiating ES cells, but cotreatment with IFN{gamma} had little effect on TF expression (Fig. 5Go). Also in contrast to its effect on PRL secretion, IFN{gamma} induced TF expression in undifferentiated cells (Fig. 5Go).



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  		Figure 5. IFN{gamma} induces TF expression in ES cells. Primary ES cells maintained in DMEM/F12 supplemented with 2% DCC-FBS were untreated (control) or treated with 8-bromo-cAMP (0.5 mM), MPA (1 µM), IFN{gamma} (1 ng/ml), or a combination of these treatments for 48 h. Protein was extracted from the cytosolic compartment, resolved by SDS-PAGE (protein loaded, 15 µg), and blotted on a polyvinylidene difluoride membrane. TF protein expression was detected using rabbit polyclonal anti-TF IgG at a concentration of 0.5 µg/ml. Protein bands were visualized by incubation with the secondary antibody, peroxidase-conjugated antirabbit IgG (developed in goat) at 1 µg/ml, followed by detection by enhanced chemiluminescence. *, P = 0.001–0.01 compared with cells transfected with 12S-E1A.

 
Spatial expression of TF protein in the human endometrium
The induction of TF by cAMP, but apparent failure of inhibition by IFN{gamma}, 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. 6AGo). However, moderate staining was also apparent in the basal stromal compartment near the endometrial-myometrial junction (Fig. 6BGo). 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. 6CGo). 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. 6DGo).



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  		Figure 6. Immunohistochemical localization of TF in human endometrium. A, Late secretory phase endometrium showing prominent TF immunostaining in the endometrial surface epithelium (arrow) and the stromal compartment in the upper part of the superficial endometrial layer. B, Higher magnification of the same section illustrating the sharp transition between TF-positive surface epithelial cells and TF-negative glandular epithelial cells (arrow). C, Late secretory phase endometrium showing moderate TF expression in the stromal compartment near the endometrial-myometrial junction. D, Intense TF immunostaining in menstrual phase endometrium. Original magnification: A, C, and D, x40; and B, x150.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There is growing evidence that the highly coordinated expression of decidual PRL in the latter half of the menstrual cycle is regulated by convergent signaling pathways that control the formation of a transcriptionally active complex containing at least C/EBPß, nuclear receptors, and associated coactivators (13, 20). Here we demonstrate that activation of the IFN{gamma}-signaling pathway antagonizes PKA-dependent decidual PRL gene expression in differentiating ES cells through inhibition of transcription. This response to IFN{gamma} 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{gamma}-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{gamma} 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{gamma}-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{gamma}-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{gamma} 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{gamma} 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-{alpha} (38, 39, 40, 41, 42, 43). Second, only a few studies have examined IFN{gamma} protein expression in the nonpregnant human uterus, and although there is agreement that IFN{gamma} immunoreactivity does not vary with the stage of the cycle, no consensus exists on its spatial expression or the identity of IFN{gamma}-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{gamma} in the human endometrium. Yeaman et al. (6), using a culture system of fresh uterine sections, found no IFN{gamma} 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{gamma} 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{gamma} failed to antagonize the gross morphological transformation of spindle-shaped undifferentiated ES cells into rounded decidual cells (data not shown). Furthermore, IFN{gamma} did also not repress TF expression in response to cAMP treatment. Together these observations indicate that endometrial IFN{gamma} 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{gamma} 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{gamma}, Stat-1 cooperates with Sp-1 in the regulation of certain target promoters (23). Furthermore, IFN{gamma} 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{gamma} 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{gamma} is gene specific. The potent inhibition of PRL expression provides a paradigm that illustrates the capacity of IFN{gamma} to modulate ES cell decidualization. In addition, IFN{gamma} 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.


   Acknowledgments
 
We are indebted to Dr. Birgit Gellersen, Institute for Hormone and Fertility Research (Hamburg, Germany); Dr. Pierre Chambon, INSERM (Strasbourg, France); Drs. Ian Kerr, Nick Jones, and Malcolm Parker, Imperial Cancer Research Fund (London, UK); Dr. Richard Maurer, University of Oregon (Portland, OR); and Dr. Bernd Groner, Institute for Biomedical Research (Frankfurt, Germany), for their generous gifts of plasmids and other reagents.


   Footnotes
 
1 This work was supported by Wellcome Trust Clinician Scientist Fellowship 54043 (to J.J.B.). Back

Received December 7, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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