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Molecular Endocrinology, doi:10.1210/me.2003-0467
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Molecular Endocrinology 18 (8): 1988-1999
Copyright © 2004 by The Endocrine Society

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)

Georgia Zoumpoulidou, Marius C. Jones, Silvia Fernandez de Mattos, Julia M. Francis, Luca Fusi, Yun Soo Lee, Mark Christian, Rana Varshochi, Eric W.-F. Lam and Jan J. Brosens

Institute of Reproductive and Developmental Biology (G.Z., M.C.J., J.M.F., L.F., Y.S.L, M.C., J.J.B), Wolfson & Weston Research Centre for Family Health and Cancer Research-UK Labs and Section of Cancer Cell Biology (S.F.d.M., R.V., E.W.-F.L.), Department of Cancer Medicine, Imperial College London, Faculty of Medicine, Hammersmith Hospital, London W12 0NN, United Kingdom

Address all correspondence and requests for reprints to: Dr. Jan Brosens, Institute of Reproductive and Developmental Biology, Wolfson & Weston Research Centre for Family Health, Imperial College London, Faculty of Medicine, Hammersmith Hospital, London W12 0NN, United Kingdom. E-mail: j.brosens{at}imperial.ac.uk.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
All cardinal events during the reproductive cycle, including ovulation, implantation, and menstruation, are characterized by a profound tissue remodeling and an associated local inflammatory response. The ovarian hormone progesterone is a key modulator of inflammatory signals in reproductive tissues, but the underlying mechanisms are not well understood. In this study, we report that differentiating human endometrial stromal cells (ESCs) acquire resistance to interferon-{gamma} (IFN{gamma})-dependent signal transducers and activators of transcription (STAT) 1 signaling, although phosphorylation, nuclear translocation, and binding of STAT1 to DNA, are unaffected. These observations prompted an investigation into the role of nuclear repressors of STAT1 signaling. We demonstrate that protein inhibitor of activated STAT-y is complexed to the progesterone receptor (PR) in human ESCs and that its ability to repress STAT1 signaling is dependent upon activation of PR in response to hormone binding. Conversely, IFN{gamma} and protein inhibitor of activated STAT-y synergistically inhibited PR-dependent transcription, demonstrating that the progesterone and IFN{gamma} signaling pathways engage in reciprocal transcriptional antagonism in human endometrium.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
OVARIAN PROGESTERONE IS the key hormonal regulator of immune responses in the female reproductive tract. All major reproductive events in the ovary and uterus, including ovulation, implantation, decidualization, luteolysis, menstruation, and parturition, are characterized by influx of distinct immune cells, expression of proinflammatory cytokines, and profound tissue remodeling. The effects of progesterone on inflammatory mediators are complex and involve modulation of local chemokine and cytokine production, direct effects on immune cell function, and attenuation of the inflammatory signaling pathways activated in target cells (1, 2, 3, 4, 5, 6). In humans, the postovulatory rise in progesterone levels induces expression of macrophage inflammatory protein-1ß and IL-15 by endometrial stromal cells (ESCs), which in turn provide the chemotactic and proliferative signals for the recruitment of CD56+, CD16– uterine natural killer (uNK) cells (1, 2). Influx of uNK cells in the early secretory phase of the cycle coincides with an acute increase in endometrial interferon-{gamma} (IFN-{gamma}) expression, a T helper 1-type cytokine strongly implicated in effecting vascular remodeling necessary for embryo implantation and subsequent placenta formation (3, 7, 8, 9). However, persistently elevated peripheral and local IFN{gamma} levels are associated with a spectrum of pregnancy disorders, including recurrent miscarriages, pre-eclampsia, and fetal growth retardation (10, 11, 12). Moreover, IFN{gamma} inhibits full differentiation of ESCs (decidualization) in vitro and induces apoptosis in trophoblast cultures (13, 14). These observations suggest that tissue homeostasis in the endometrium during the secretory phase of the cycle and in pregnancy is dependent upon a balance between proinflammatory actions of progesterone and compensatory antiinflammatory responses.

The cellular responses to IFN{gamma} are mediated predominantly through activation of the Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) (15, 16). Binding of IFN{gamma} to its receptor results in activation of JAK1 and JAK2, which phosphorylate the latent cytoplasmic transcription factor STAT1. STAT1 is then translocated into the nucleus where it binds to IFN{gamma} activation site (GAS), a response element found in the promoter region of numerous IFN{gamma}-inducible genes (15, 17). STAT1 signal transduction can be subject to negative regulation in the cytoplasm through a variety of mechanisms, including receptor degradation, inhibition of JAK activity by suppressor of cytokine signaling family of proteins, and several protein tyrosine phosphatases capable of dephosphorylating either cytokine receptors, JAKs, or STATs. In the nucleus, STAT1 signaling can be further attenuated by specific phosphatases such as TC45 and by members of the family of protein inhibitor of activated STAT (PIAS) (18, 19, 20, 21, 22, 23, 24).

In mammalian cells, the PIAS family consists of five multifaceted proteins (PIAS1, PIAS3, PIASx{alpha}, PIASxß, and PIASy) capable of functioning as transcriptional coregulators, E3-type small ubiquitin-like modifier (SUMO) ligases, and modulators of chromosome structure (25, 26, 27, 28, 29). PIAS1 and PIASy have both been shown to antagonize STAT1 signaling through distinct mechanisms. Upon IFN{gamma} stimulation, PIAS1 binds to the activated STAT1 dimer and inhibits gene activation by interfering with the DNA binding activity of STAT1 (23). In contrast, PIASy does not block STAT1 DNA binding activity but is capable of recruiting histone deacetylases (HDACs) (24, 30). Recent evidence suggests that PIASy also antagonizes other transcription factors, including the activated androgen receptor (AR), p53, Smads, lymphoid-enhancing factor 1, and the orphan nuclear receptor Nur-related factor 1 (30, 31, 32, 33, 34). Here we report that PIASy is complexed to progesterone receptor (PR) in human ESCs. Moreover, we provide evidence suggesting a role for PIASy in integrating progesterone and IFN{gamma} signaling pathways in differentiating human endometrium.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
IFN{gamma}-Dependent Gene Expression Is Repressed upon ESC Differentiation
Progesterone-mediated ESC differentiation is dependent upon elevated intracellular cAMP levels and sustained activation of the protein kinase A pathway (35). In human endometrium, activated protein kinase A induces the expression and activation of several transcription factors, including CCAAT/enhancer binding protein ß, and STAT5, capable of binding to PR (35, 36, 37, 38). In contrast, IFN{gamma} and activation of the STAT1 signaling pathway represses the expression of decidual markers such as prolactin in primary cultures made to differentiate in response to treatment with 8-bromo-cAMP and medroxyprogesterone acetate (MPA, a synthetic progestin) (13, 38). While investigating the underlying mechanism, we fortuitously observed that differentiating ESCs acquire resistance to IFN{gamma} signaling. Primary undifferentiated ESCs and decidualizing cells were transiently transfected with a luciferase reporter containing either four copies of the wild-type (wt) GAS (GAS-wt/luc) or mutant (mt) GAS (GAS-mt/luc) (39). The terms "differentiating" or "decidualizing" ESCs refer to cells pretreated with 8-bromo-cAMP and MPA for 48 h. Incubation of cultures with 0.1 ng/ml IFN{gamma} yielded a 22-fold induction in GAS-wt/luc activity in undifferentiated cells, but only a 4-fold induction in reporter activity was observed in decidualizing cells (Fig. 1AGo). Furthermore, GAS-wt/luc activity upon IFN{gamma} stimulation was lower in differentiating cultures at all concentrations tested. As expected, IFN{gamma} failed to elicit GAS-mt/luc activity. The basal GAS-mt/luc activity in undifferentiated was comparable to that in decidualizing cells, indicating equal transfection efficiency (Fig. 1BGo). This was further confirmed by Western blot analysis of undifferentiated and decidualized cells transiently transfected with a V5-tagged membrane PR-{alpha} (Fig. 1CGo).



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  		Fig. 1. Decidualization Attenuates the IFN{gamma}-Dependent GAS-Driven Transcription

A and B, Untreated primary ESC cultures or decidualizing cultures, pretreated with 0.5 mM 8-bromo-cAMP and 10–6 M MPA for 48 h, were transiently transfected with either the GAS-wt/luc or the GAS-mt/luc reporter construct. Some undifferentiating and decidualizing transfected cultures were subsequently treated for 20 h with various concentrations of IFN{gamma}, as indicated. The results show mean luciferase activity ± SD of triplicate measurements. C, Equal transfection efficiency of undifferentiating and decidualized ESCs where cultures untreated or pretreated with 0.5 mM 8-bromo-cAMP and 10–6 M MPA for 48 h were transfected with the indicated amounts of V5-mPR expression vector. After 20 h, whole cell lysates were subjected to Western blot analysis using antibody against the V5 epitope with ß-actin probing serving as loading control.

 
Next we investigated whether activation of endogenous genes upon IFN{gamma} treatment also differs between proliferating and decidualizing primary cultures. Real-time quantitative-PCR (RTQ-PCR) was used to determine the transcript levels of two known IFN{gamma}-inducible genes, IRF1 (IFN response factor 1) and Nmi (N-Myc interactor), in undifferentiated and in cells pretreated with 8-bromo-cAMP and MPA for 48 h. IRF1 (Fig. 2AGo) and Nmi (Fig. 2BGo) transcripts were strongly induced after 8 h of stimulation with IFN{gamma}, in a dose-dependent manner. However, expression levels in decidualized cells were markedly lower when compared with undifferentiated cells. Interestingly, the basal expression level of Nmi, but not IRF1, was found to be consistently lower in differentiating ESCs (Fig. 2BGo). Together, these observations demonstrate that decidual transformation of human ESCs results in relative resistance to IFN{gamma} signaling, characterized by an attenuated GAS-mediated transcriptional response.



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  		Fig. 2. Endogenous IFN{gamma}-Inducible Genes Expression Is Attenuated in Decidualizing ESCs

Untreated primary ESC cultures or cultures pretreated with 0.5 mM 8-bromo-cAMP and 10–6 M MPA for 48 h were stimulated with IFN{gamma} (1 or 10 ng/ml) for 8 h. Total RNA was prepared, reversed transcribed, and the expression of (A) Nmi RNA and (B) IRF1 RNA was analyzed by real-time PCR, and normalized to the level of GAPDH. The data shown are representative of three independent experiments.

 
Decidualization Does Not Interfere with the STAT1 Signal Transduction or DNA Binding
Activation of STAT1 in response to IFN{gamma} involves posttranslational modifications such as phosphorylation of residues Tyr701 and Ser727 (16, 40). To determine whether the decidual process interferes with STAT1 signal transduction, we examined the induction of total STAT1 and phospho-STAT1 after 8 h of stimulation with IFN{gamma} in undifferentiated and differentiating cells. Western blot analysis of cytoplasmic and nuclear protein fractions were separated by SDS-PAGE and probed sequentially with antiserum against phospho-STAT1 and total STAT1. As previously described, the IFN{gamma}-induced STAT1 phosphorylation was accompanied by an up-regulation of STAT1 expression in both proliferating and decidualized cells (Fig. 3AGo) (13). However, the total STAT1 levels and nuclear accumulation of phospho-STAT1 upon treatment with IFN{gamma} remained unaltered by decidualization. Furthermore, exogenously expressed STAT1 enhanced IFN{gamma}-induced GAS-wt/luc activity in proliferating cells in transient transfection experiments but not in differentiating cultures (data not shown), underscoring the notion that the relative resistance to IFN{gamma} signaling in differentiating cells is not due to limited amount of STAT1.



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  		Fig. 3. STAT1 Phosphorylation, Nuclear Translocation, and DNA Binding Ability Are Not Impaired in Decidualized ESCs

A, Untreated primary ESC cultures or cultures pretreated with 0.5 mM 8-bromo-cAMP and 10–6 M MPA for 48 h were stimulated with IFN{gamma} (1 or 10 ng/ml) for 8 h or were left unstimulated. Nuclear and cytoslic extracts were prepared and subjected to Western blot analysis. B, Nuclear proteins from undifferentiated ESCs (lanes 1 and 2), undifferentiated ESCs treated with 1 ng/ml IFN{gamma} for 8 h (lanes 3 and 4), decidualizing cells (pretreated with 0.5 mM 8-bromo-cAMP and 10–6 M MPA for 48 h; lanes 5 and 6), and decidualizing cells treated with 1 ng/ml IFN{gamma} for 8 h (lanes 7 and 8) were incubated with labeled probe containing the consensus STAT1 binding site for EMSA. Competition was carried out by addition of 100-fold excess of unlabeled probe. In the absence of IFN{gamma}, two specific complexes were apparent (indicated by arrows). However, a third protein-DNA complex formed in both undifferentiated and decidualizing cells treated with IFN{gamma} (indicated by asterisks). To determine whether this IFN{gamma}-induced complex represent STAT1, nuclear proteins from decidualizing cultures treated with 1 ng/ml IFN{gamma} were resolved on a separate gel for supershift analysis (lanes 10–12). The presence of antiserum to STAT1 blocked the formation of the IFN{gamma}-inducible protein-DNA complex.

 
Next we examined the possibility that decidual transformation of ESCs interferes with the DNA binding activity of STAT1. Nuclear extracts were prepared from primary undifferentiated and differentiating cells treated with or without IFN{gamma} for 8 h and analyzed by EMSA using the GAS element as probe. Under all culture conditions, two protein-DNA complexes were apparent (Fig. 3BGo). IFN{gamma} treatment increased the intensity of these two complexes and induced a third complex that migrated between the two constitutive bands. These IFN{gamma}-dependent effects are likely to be mediated by STAT1 because incubation of nuclear extracts with anti-STAT1 antibody not only blocked the formation of the IFN{gamma}-inducible band but also lowered the intensity of the two constitutive bands (Fig. 3BGo). Notably, the DNA binding complexes in differentiating and proliferating cells were indistinguishable. Together, these results demonstrate that the attenuated IFN{gamma} response in decidualizing cells is not a consequence of impaired STAT1 activation, nuclear accumulation of phospho-STAT1, or the formation of distinct DNA-bound protein complexes.

Nmi Fails to Enhance IFN{gamma}-Dependent Transcription in Decidualized Cells
We reasoned that down-regulation of Nmi expression could possibly account for the reduced IFN{gamma} responses in differentiating cells (Fig. 2BGo). Nmi interacts with all STATs, except STAT2, and has been shown to potentiate IFN{gamma}-dependent transcription by facilitating recruitment of CBP/p300 coactivator proteins to activated STAT1 (41). Confocal microscopy studies revealed that Nmi protein is indeed expressed in undifferentiated ESCs, predominantly in the perinuclear cytoplasm (Fig. 4AGo, upper panel). In agreement with the RTQ-PCR data (Fig. 2Go), treatment of cells with 8-bromo-cAMP and MPA for 48 h markedly lowered Nmi expression (Fig. 4AGo, lower panel and data not shown). In transient transfection experiments, expression of Flag-tagged Nmi enhanced IFN{gamma}-induced GAS-wt/luc activity in undifferentiated cells, from 19- to 28-fold, but had remarkably little or no effect on reporter activity in decidualizing cells (Fig. 4BGo). In these experiments, Flag-Nmi was efficiently expressed in decidualizing cells as determined by Western blot analysis of parallel transfected cultures using an anti-Flag antibody (data not shown).



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  		Fig. 4. Compensation for the Reduced Nmi Expression in Decidualized ESCs through Its Overexpression Fails to Relieve the Attenuated IFN{gamma} Response

A, Primary ESCs plated in chamber slides were maintained in culture medium supplemented with 2% DCC-FBS and received no further treatment or were stimulated with 0.5 mM 8-bromo-cAMP and 10–6 M MPA. Confocal microscopy demonstrates endogenous Nmi immunoreactivity in perinuclear cytoplasm of untreated ESCs (upper panels). However, upon treatment with 8-bromo-cAMP and MPA for 2 d, Nmi immunoreactivity was barely detectable (bottom panels). B, Undifferentiated ESC cultures or decidualizing cultures, pretreated with 0.5 mM 8-bromo-cAMP and 10–6 M MPA for 48 h, were transiently transfected with the GAS-wt/luc reporter construct and the empty pSG5 control vector or pSG5-Nmi expression vector. Some undifferentiating and decidualizing transfected cultures were subsequently treated with 1 ng/ml IFN{gamma} for 20 h. The results show mean luciferase activity ± SD of triplicate measurements. dapi, 4',6-Diamidino-2 phenylindole.

 
Decidualization Enhances PIASy Transrepression
The failure of exogenously expressed Nmi to enhance or restore reporter activity in differentiating cells, despite normal STAT1 activation, suggested that IFN{gamma}-dependent transcription may be actively repressed. PIAS1 and PIASy are two nuclear proteins capable of inhibiting STAT1 transactivation (23, 24). The results outlined above strongly suggested a role for PIASy that, in contrast to PIAS1, blocks STAT1-mediated transcription without affecting its DNA binding activity. We used Western blot analysis of whole cell extracts to examine whether PIASy expression is regulated upon decidualization. As shown in Fig. 5AGo, decidualization was not associated with increased PIASy expression levels. Instead, a modest down-regulation was apparent and was further enhanced in the presence of IFN{gamma}. This raised the possibility that decidual transformation of human ESCs induces an as yet uncharacterized STAT1 corepressor. Alternatively, the apparent down-regulation of PIASy in differentiating cells could reflect turnover of activated protein. To differentiate between these possibilities, undifferentiated and decidualizing cells were transiently transfected with GAS-wt/luc and increasing amounts of an expression vector encoding PIASy. Although exogenous PIASy repressed basal GAS-wt/luc activity in both undifferentiated and decidualizing cells, it had surprisingly little effect on IFN{gamma}-induced promoter activity in proliferating ESCs (Fig. 5BGo). In fact, the effect of exogenously expressed PIASy varied between proliferating cultures and either no effect, modest inhibition, or even stimulation of IFN{gamma}-dependent GAS-wt/luc activity was observed (data not shown). In contrast, PIASy consistently inhibited IFN{gamma}-induced GAS-wt/luc activity in differentiating cells, indicating that PIASy repressive activity is markedly enhanced upon pretreatment with 8-bromo-cAMP and MPA (Fig. 5BGo). In 293T cells, repression of STAT1-mediated gene activation has been shown to be dependent on a LXXLL coregulator signature motif located at the N terminus of PIASy (24). It has also been suggested that the ring domain in PIASy, which confers SUMO E3 ligase activity, is important for transcriptional repression (30). However, in differentiating human ESC, transient transfection of mt PIASy expression vectors, deficient in either LXXLL motif (PIASy-AA) or ring domain (PIASy-RING), inhibited the IFN{gamma} response as effectively as wt PIASy (Fig. 5CGo). Together, these results indicate that the transrepression activity of PIASy is enhanced upon decidualization of primary ESC cultures through a mechanism that neither involves its amino-terminal LXXLL motif nor SUMO E3 ligase activity.



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  		Fig. 5. PIASy-Mediated Transcriptional Repression Is Enhanced upon ESC Decidualization

A, PIASy is expressed in undifferentiated and decidualizing ESCs. Undifferentiated primary ESC cultures or cultures pretreated with 0.5 mM 8-bromo-cAMP and 10–6 M MPA for 48 h were stimulated with IFN{gamma} (1 ng/ml) for 8 h. The expression of PIASy (56 kDa) was analyzed in whole cell lysates by Western blotting. B, transiently transfected PIASy efficiently inhibits IFN{gamma}-induced GAS-wt/luc activity in decidualizing ESCs. Proliferating ESC cultures or decidualizing cultures, pretreated with 0.5 mM 8-bromo-cAMP and 10–6 M MPA for 48 h, were transiently transfected with the GAS-wt/luc reporter construct as well as various amounts of pSG5-PIASy expression vector (0, 40, 100, and 250 ng/well). The empty vector pSG5 was used as filler. C, Additional undifferentiated ESCs or decidualizing cells were also transiently transfected with GAS-wt/luc and either pSG5 or expression vectors encoding for wt-PIASy (hPIASy), PIASy with a mutated LXXLL motif (PIASy-AA), or an E3-SUMO ligase-deficient PIASy mutant (PIASy-RING). Cultures subsequently received no additional treatment or were treated with 1 ng/ml IFN{gamma} for 20 h. The results show mean luciferase activity ± SD of triplicate measurements.

 
PR Interacts with PIASy in Human ESCs
Physical interaction between PIASy and the AR have been shown to attenuate the transcriptional responses to androgens in prostate cancer cells (31). In analogy with these observations, we postulated that interaction between PIASy and PR may integrate IFN{gamma} and progesterone signaling in human endometrium. To determine whether PIASy binds to PR, His epitope-tagged PR was transfected with or without a T7-tagged PIASy in primary ESC cultures with no treatment or stimulated with 10–6 M MPA. His-PR was precipitated using nickel nitrilotriacetic acid (Ni-NTA) beads followed by immunoblotting with anti-T7 antibody. As shown in Fig. 6AGo, T7-PIASy interacted with His-PR in a manner relatively insensitive to the presence of MPA. Various members of the PIAS family of proteins, including PIAS1, PIASx{alpha}, and PIASy, have been shown to interact with the DNA binding domain (DBD) of AR (31, 42, 43). Because the DBDs of PR and AR are highly homologous, we used an in vitro binding assay to test whether the DBD of PR could mediate the interaction with PIASy. Glutathione-S-transferase (GST) or GST-bound DBD of PR (GST-PR-DBD) were bound to glutathione-agarose beads and mixed with 35S-labeled PIASy. 35S-labeled FoxO1a protein was used as control. A specific interaction between PR-DBD and PIASy, but not with FoxO1a, was observed (Fig. 6BGo).



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  		Fig. 6. PIASy-PR Interaction Is Ligand Independent in Vitro and in Vivo

A, In vivo association of PR and PIASy. Primary ESCs were transfected with an expression vector encoding N-terminally HIS-tagged PR, T7 epitope-tagged PIASy, or both. Subsequently, cultures remained either untreated or were stimulated with 10–6 M MPA for 48 h. Equivalent amounts of total cellular proteins were precipitated using Ni-NTA agarose beads and then analyzed by immunoblotting with T7-tag monoclonal antibody. B, PIASy binds to the isolated DBD of PR. In vitro-translated 35S-labeled PIASy or Foxo1a were incubated with either GST-fused DBD of PR or GST alone immobilized on glutathione-agarose beads as indicated. The bound proteins were resolved on 10% SDS-polyacrylamide gels and visualized by autoradiography. C, Endogenous PIASy colocalizes with PR in nuclear bodies. The intracellular distribution of endogenous PIASy and PR was detected by indirect immunofluorescence and analyzed by confocal microscopy in untreated primary ESCs and in cells treated with 10–6 M MPA for 48 h. PIASy, detected with anti-PIASy antibody, localized in punctate nuclear bodies in the absence (upper panels) or presence (lower panels) of MPA. PR immunofluorescence showed a more homogeneous distribution throughout the nucleus although discrete punctate structures were also apparent and partially overlapped with PIASy nuclear bodies. Treatment with MPA did not elicit an apparent redistribution of PR or PIASy.

 
These observations demonstrate that transfected PIASy and PR physically interact in human ESCs. In an endogenous context, we performed double color immunofluorescence experiments on untransfected cells that were either untreated or treated with MPA for 48 h. PIASy has been shown to reside predominantly in distinct punctate nuclear structures in transfected COS-7 cells (33). Confocal microscopy revealed a similar pattern of focal nuclear staining for endogenous PIASy in human ESCs (Fig. 6CGo). In contrast to other steroid receptors, both unliganded and activated PRs reside in the nucleus of human ESCs. Although PR immunofluorescence was generally diffuse in the nucleus, small discrete foci were also apparent. Overlay of the images revealed that PR and PIASy foci colocalized, although the PR punctate structures were invariably smaller than the PIASy nuclear bodies. Treatment of cultures with MPA did not result in an apparent redistribution of either PR or PIASy (Fig. 6CGo).

PR-PIASy Interaction Integrates IFN{gamma} and Progesterone Signaling Pathways
Although our data suggest that PIASy interacts with PR in a predominantly ligand-independent manner, hormone binding could be critical for the attenuated transcriptional response to IFN{gamma} upon decidualization. To test this hypothesis, we first examined whether PR modulates GAS-wt/luc activity in undifferentiated ESCs. Primary cultures were transiently transfected with the reporter construct and expression vectors encoding either for PR-A, PR-B, or the isolated DBD of PR coupled to the nuclear localization signal (DBD-NLS). Subsequently, cells remained untreated or were stimulated with IFN{gamma}, MPA, or both. In the absence of overexpressed PR, IFN{gamma}-induced reporter activity was attenuated by cotreatment with MPA (Fig. 7AGo), albeit to a lesser extent than observed in decidualizing cultures pretreated with 8-bromo-cAMP and MPA. Remarkably, transfection of either PR-A, PR-B, or DBD-NLS elicited GAS-wt/luc activity in untreated cells. This ability of exogenously expressed wt PR to transactivate GAS-wt/luc in the absence of IFN{gamma} was abolished upon addition of MPA to the cultures. Moreover, the inhibitory effect of MPA on IFN{gamma}-dependent reporter activity was greatly enhanced in the presence of coexpressed PR-A or PR-B but not by transfected DBD-NLS (Fig. 7AGo). The results establish that liganded PR represses IFN{gamma}-dependent transcriptional responses, whereas unliganded PR may modulate the basal expression of IFN{gamma}-target genes, probably through squelching of endogenous repressor(s). Because expression of PIASy inhibited GAS-wt/luc activity in the absence of activated STAT1 (Fig. 5BGo), it is tempting to speculate that it may also play a role in the active repression of basal promoter activity in human endometrial cells.



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  		Fig. 7. PIAS-PR Complex Integrates the Progesterone and IFN{gamma} Signaling Pathways

A, PR represses IFN{gamma}-mediated transcription in a ligand-dependent manner. Undifferentiated ESC cultures were transiently transfected with the GAS-wt/luc reporter construct and the empty vector pSG5 or expression vectors encoding for PR-A, PR-B or the DBD of PR coupled to the nuclear localization signal (DBD-NLS). Some cultures remained untreated, whereas others were treated for 20 h with 10–6 M MPA, 1 ng/ml IFN{gamma}, or a combination of both. B, Overexpressed PR, AR, and ER induce GAS activity in the absence of ligand. Undifferentiated ESC cultures were transiently transfected with the GAS-wt/luc reporter construct and the empty vector pSG5 or expression vectors encoding either for GR, PR-A, AR, or ER. In a parallel experiment the GAS-wt/luc was replaced by PRE/–32/luc3. Transfected cultures were left untreated or stimulated with the appropriate ligand (10–6 M dexamethasone, 10–6 M MPA, 10–6 M DHT, or 10–8 M E2, respectively) for 20 h. C, The DBD of PR blocks PIASy-mediated transcriptional repression. Decidualizing cultures, pretreated with 0.5 mM 8-bromo-cAMP and 10–6 M MPA for 48 h, were transiently transfected with GAS-wt/luc and PIASy as well as various amounts of DBD-NLS of PR (0, 25, 75, and 150 ng/well). The empty vector pSG5 was used as filler. As indicated, some cultures were treated with 1 ng/ml IFN{gamma} for 20 h. The decidualizing stimulus was maintained throughout the experiment. D, IFN{gamma} and PIASy antagonize PR-mediated transcriptional response. Undifferentiated ESC cultures were transiently transfected with the GAS-wt/luc, PR-B, and the empty vector pSG5 or pSG5-PIASy. Subsequently, the cultures remained untreated or were stimulated for 20 h with 10–6 M MPA, alone or in combination with 1 or 0.1 ng/ml IFN{gamma}. The results show mean luciferase activity ± SD of triplicate measurements.

 
The ability of unliganded PR to activate GAS-dependent transcription was surprising and, to the best of our knowledge, has not yet been reported for other nuclear steroid receptors. To determine whether this is a feature unique to PR, primary cultures were transiently transfected with GAS-wt/luc and expression vectors encoding for GR, PR-B, AR, or ER{alpha}. Cultures either remained untreated or were treated with the appropriate ligand. Figure 7BGo shows that not only PR but also AR and, albeit to a lesser extent, ER enhanced basal reporter activity in human ESCs in the absence of ligand. In contrast, unliganded GR had no apparent effect on GAS-wt/luc activity, although treatment of cultures with dexamethasone modestly enhanced reporter activity. Parallel cultures were transiently transfected with a synthetic promoter construct consisting of two palindromic progesterone response elements (PREs) upstream of the minimal decidual prolactin promoter (PRE/–32/luc) (36). As expected, transfection of either GR, AR, PR, but not ER, resulted in strong reporter activity in an entirely ligand-dependent manner (Fig. 7BGo).

We postulated that if the activation of PR plays an integral role in enhancing PIASy repressive activity, then overexpression of the isolated DBD-NLS of PR, which interacts with PIASy but lacks a ligand binding domain, should reverse the inhibitory effect of PIASy on GAS-wt/luc activity in differentiating ESCs. Figure 7CGo shows that this is indeed the case. Decidualizing ESCs, pretreated with 8-bromo-cAMP and MPA for 48 h, were transiently transfected with the GAS-wt/luc reporter. Cotransfection of PIASy markedly inhibited the IFN{gamma}-dependent GAS-wt/luc activity, but this was entirely reversible by increased expression of DBD-NLS (Fig. 7CGo).

Next, we examined whether IFN{gamma} interferes with the transcriptional responses of activated PR. Primary cultures were transfected with an expression vector for PR-B and PRE/–32/luc. Treatment of cultures with MPA yielded a 38-fold increase in promoter activity (Fig. 7DGo). However, the transactivation potential of activated PR was markedly reduced when cells were cotreated with IFN{gamma}. Transiently transfected PIASy also attenuated the transcriptional response to MPA. Moreover, PIASy synergized with IFN{gamma} in antagonizing PR-dependent transcription (Fig. 7DGo).

Antagonism between Progesterone and IFN{gamma}-Signaling Pathways Is Not Reversible by Trichostatin A (TSA)
Recent studies have shown that PIASy can interact with HDAC1 and HDAC2 and that trichostatin A (TSA, an HDAC inhibitor) reverses the inhibitory effect of PIASy on AR- (44) and Smad-dependent transcription (30). Although HDACs are widely involved in gene repression, multiple HDAC-independent mechanisms of repression exist (45, 46, 47). Furthermore, the precise mechanism of inhibition used by a given corepressor can vary, depending on promoter context and cell type. We tested whether treatment with TSA could restore sensitivity to IFN{gamma} signaling in decidualizing ESCs. Primary undifferentiated and cells pretreated with 8-bromo-cAMP and MPA were transfected with GAS-wt/luc. To account for possible nonspecific transcriptional effects of TSA, parallel cultures were transfected with GAS-mt/luc, and the results are presented as the fold induction of GAS-wt/luc divided by the fold induction of GAS-mt/luc (Fig. 8AGo). After transfection, the cultures remained either untreated or were stimulated with 1 ng/ml IFN{gamma} and various concentrations of TSA for 20 h. In proliferating cells, TSA at low concentrations (≤60 nM) increased IFN{gamma}-induced GAS-wt/luc activity but paradoxically inhibited reporter activity at higher concentrations (≥125 nM). Differentiating ESCs were less sensitive to effects of TSA, although a modest increase in IFN{gamma}-induced GAS activity was apparent. The results indicate that repression of STAT1-dependent transcription in differentiating ESCs is only partially relieved by TSA, indicating possible involvement of HDAC-independent as well as HDAC-dependent mechanisms of repression. Notably, IFN{gamma}-induced reporter activity in proliferating and decidualizing cells was comparable in the presence of 250 nM of TSA, but this could be accounted for by the paradoxical inhibition of GAS-wt/luc activity in proliferating cells. Using a similar experimental approach, we found that the inhibitory effects of IFN{gamma} upon PR-dependent activation of PRE/–32/luc are also relatively insensitive to TSA treatment and, hence, it is likely to involve TSA-insensitive HDACs or an HDAC-independent mechanism (Fig. 8BGo).



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  		Fig. 8. HDAC Inhibitor TSA Does Not Completely Eliminate the PR-IFN{gamma} Antagonism

A, Undifferentiated or decidualizing cultures, pretreated with 0.5 mM 8-bromo-cAMP and 10–6 M MPA for 48 h, were transiently transfected with 0.4 µg GAS-wt/luc or GAS-mt/luc. Some cultures were treated with 1 ng/ml IFN{gamma} and the indicated amounts of TSA. The decidualizing stimulus was maintained throughout the experiment. Fold inductions were calculated as the mean luciferase activity in each culture divided by the mean luciferase activity of cultures not treated with IFN{gamma} and TSA. This ratio was set as 1 for the control cultures. The GAS-wt/luc fold induction was corrected against the fold induction of the nonresponsive GAS-mt/luc. B, ESC cultures were transiently transfected with 0.4 µg PRE/–32/luc or –32/luc together with 0.1 µg pSG5-PRB expression vector. Some cultures were treated with 1 ng/ml IFN{gamma}, 10–6 M MPA and TSA, as indicated, for 20 h. Results show fold induction, calculated as the mean luciferase activity in each culture divided by the mean luciferase activity of cultures not treated with MPA and TSA. This ratio was set as 1 for the control cultures. The PRE/–32/luc reporter fold induction was corrected against the fold induction of the minimal –32/luc construct.

 
In summary, we demonstrated that the progesterone and IFN{gamma} signaling pathways engage in mutual transcriptional antagonism in human ESCs. The observations that PIASy interacts with PR in a ligand-independent manner and that both proteins colocalize in distinct subnuclear structures provide a potential point of convergence for these two signal transduction pathways. However, the mechanism whereby progestins modulate the ability of PIASy to inhibit STAT1-dependent transcription requires further exploration. One possible mechanism is that hormone-binding induces a configurational change in the PR-PIASy complex, allowing recruitment of specific corepressors, such as receptor-interacting protein 140, ligand-dependent corepressor, or C-terminal binding proteins, capable of inhibiting transcription in a HDAC-independent manner (45, 46, 47, 48). Conversely, the formation of such a complex may be facilitated by activated STAT1, which could account for the synergistic inhibitory effect of IFN{gamma} and PIASy on PR-dependent transcription. Alternatively, critical corepressor(s) could be either induced or posttransciptionally modified upon ESC differentiation. For instance, emerging evidence indicates that decidual transformation of ESCs is associated with profound alterations in the SUMO conjugation pathway (data not shown) and the ability of a variety of nuclear proteins, including PR, STAT1, HDACs, C-terminal binding proteins, and histones, to control gene expression is profoundly altered upon targeted sumoylation (28, 49, 50, 51, 52, 53, 54). Finally, we cannot exclude the possibility that the transcriptional antagonism between progesterone and IFN{gamma} signaling pathways also involves competition for limited amounts of common cofactor. For example, competition for CBP/p300 has been shown to underpin IFN{gamma}/STAT1-mediated repression of activator protein-1/ets-dependent transcription (55).

From a clinical viewpoint, progestins are widely used in the treatment of a variety of reproductive disorders, including menstrual disturbances, endometriosis, recurrent miscarriages, and preterm labor. These common disorders are all characterized by influx of immune cells in uterine tissues and a local inflammatory response. This study demonstrates that some of the antiinflammatory actions of progestins in human endometrium involve antagonism of IFN{gamma}-mediated gene expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Primary Culture
ESCs 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 Eagle’s buffered saline containing 100 U/ml penicillin and 100 µg/ml streptomycin. After enzymatic digestion, the stromal cells were separated from epithelial cells, and passed into culture as described previously (36, 42). Proliferating ESCs were cultured in maintenance medium of DMEM/F-12 containing 10% dextran-coated charcoal-treated fetal bovine serum (DCC-FBS) and 1% antibiotic-antimycotic solution. Confluent monolayers were treated in DMEM/F-12 containing 2% DCC-FBS with 0.5 mM 8-bromo-cAMP and 10–6 M MPA for 48 h to induce a differentiated phenotype. All experiments were carried out before the fourth cell passage.

RNA Isolation and RTQ-PCR
Total RNA was isolated using STAT-60 (Tel-Test, Friendswood, TX) and deoxyribonuclease I treated. Equal amounts of total RNA (2 µg) were reverse transcribed using the Superscript First-Strand Synthesis System for RT-PCR (Invitrogen Life Technologies, Carlsbad, CA) and the resulting first-strand cDNA was diluted and used as template in the RTQ-PCR analysis. Detection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Nmi, and IRF1 expression was performed with SYBR Green Master Mix (Applied Biosystems, Foster City, CA) and an ABI PRISM 7700 Sequence Detection System (Applied Biosystems), using the relative standard curve method. GAPDH represents a nonregulated gene and its expression served as internal control and was used to normalize for variances in input cDNA. All measurements were performed in triplicate. The following gene-specific primer pairs were designed using the ABI Primer Express software: GAPDH-sense (5'-ATTTGGTCGTATTGGGCGCCTGGTCACC-3') and GAPDH-antisense (5'-GAAGATGGTGATGGGATTTC-3'); Nmi-sense (5'-CGCGTGGACTATGACAGACAGT-3') and Nmi-antisense (5'-AAATCTTGTCAGCCACTCCAATCT-3'); IRF1-sense (5'-CATGGCTGGGACATCAACAAG-3') and IRF1-antisense (TTTGTATCGGCCTGTGTGAATG-5'). Specificity of each primer was determined using National Center for Biotechnology Information basic local alignment and search tool module.

Western Blotting and NTA Precipitation
Western blotting was performed on whole cell extracts prepared by lysing cells in Nonidet P-40 (NP-40) lysis buffer [1% NP-40, 100 mM NaCl, 20 mM Tris-HCl (pH 7.4), 10 mM NaF, 1 mM sodium orthovanadate, 30 mM Naß-glycerophosphate, and protease inhibitors] ["Complete" protease inhibitor cocktail, as instructed by the manufacturer (Roche Applied Science, Penzberg, Germany)] on ice for 15 min. Nuclear extracts were obtained using the modified method of Rittenhouse and Marcus (56). Protein yield was quantified by Bio-Rad Dc protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of proteins (20 µg) were separated on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel before electrotransfer onto polyvinylidene difluoride membrane (Hybond P; Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). Even loading and transfer efficiency were confirmed by Ponceau S staining. Nonspecific binding sites were blocked by overnight incubation with 5% dried skimmed milk in Tris-buffered saline [TBS; 130 mM NaCl, 20 mM Tris (pH 7.6)]. Primary antibodies used were as follows: mouse monoclonal anti-V5 (Invitrogen Life Technologies, R960-CUS); mouse monoclonal anti-ß-actin (abcam, ab6276); mouse monoclonal anti-STAT1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; sc-464); rabbit anti-phospho-STAT1 (Upstate, Lake Placid, NY); rabbit polyclonal anti-PIASy antibody (IMGENEX, San Diego, CA; IMG-290). Blots were exposed to the primary antibody diluted 1:1000 in TBS with 5% dried nonfat milk, for 1 h at room temperature, and then incubated with secondary peroxidase conjugated goat antirabbit IgG (Sigma, St. Louis, MO), also for 1 h at room temperature. Protein bands were visualized by enhanced chemiluminescence (ECL Western Blotting Detection; Amersham Biosciences).

Precipitation experiments were carried out using cell extracts prepared by lysing His-PR and His-PR/T7-PIASy transfected cells with four times the packed cell volume of high-salt lysis buffer [300 mM NaCl, 20 mM HEPES (pH 7.9), 1 mM MgCl2, 1 mM EDTA (pH.8.0), 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM NaF, 5 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride] on ice for 30 min. The lysates (50 µg), after adjusting to 500 µl, were incubated with 40 µl (50% vol/vol) of either normal Sepharose CL-6B beads (Sigma) or Ni-NTA Agarose Beads (QIAGEN, Valencia, CA) in lysis buffer containing 50 mM imidazole (Sigma) for 2 h at 4 C. Afterward, the beads containing the precipitated complexes were washed thoroughly with lysis buffer (6 x 1 ml) and the precipitated proteins resolved by SDS-PAGE, transferred to nitrocellulose membranes and probed with anti-T7 mAb (Novagen, Madison, WI).

Confocal Immunofluorescence Microscopy
ESCs cultured on chamber slides (LabTek, Naperville, IL) were fixed in methanol and permeabilized in 0.5% Triton. Primary antibodies and dilutions were as follows: rabbit anti-Nmi (a gift from L. Naumovski, Stanford University, Stanford, CA), 1:200; rabbit anti-PIASy (IMGENEX; IMG-290), 1:250; mouse anti-PR (Novocastra, Newcastle-Upon-Tyne, UK; NCL-L-PGR-312), 1:500. Secondary antibodies used were fluorescein isothiocyanate-conjugated antirabbit antiserum, fluorescein isothiocyanate-conjugated antimouse antiserum, and tetramethylrhodamine isothiocyanate-conjugated antirabbit antiserum. Images were obtained on a Zeiss (Welwyn Garden City, Hertfordshire, UK) Meta 512 confocal microscope.

EMSA
32P-end-labeled probe, consisting of a high-affinity consensus STAT1 binding site (Santa Cruz Biotechnology), was incubated with 10 µg of nuclear protein obtained from undifferentiated and decidualizing primary ESC cultures treated with IFN{gamma} (10 ng/ml) for 6 h. Final concentrations of components in the binding reaction, including high-salt nuclear extracts, were: 15 mM HEPES, 200 mM NaCl, 5 mM MgCl2, 65 mM KCL, 0.05 mM EDTA, 0.05 mM EGTA, 1 mM dithiothreitol, 1.25 mM spermidine, 3.5% Ficoll, 0.5% NP-40, and 0.02 U poly(deoxyinosine-deoxycytosine). Specificity was determined by preincubation for 15 min on ice with a 100-fold excess of unlabeled probe, followed by addition of labeled probe and incubation for 30 min on ice. Supershift analysis was performed by adding 1 µg STAT1-antibody (Santa Cruz Biotechnology) for 60 min at 4 C before incubation with labeled probe. Protein-DNA complexes were resolved on a 4.5% polyacrylamide gel in 0.25x TBE running buffer for 2 h at 200 V. The gel was dried under vacuum for 1 h at 80 C and protein-DNA complexes were visualized by autoradiography.

Reporter Gene Constructs, Expression Vectors, and Transient Transfections
The GAS-wt/luc and GAS-mt/luc reporter constructs were obtained from K. Ozato (National Institutes of Health, Bethesda, MD). Expression vectors for wt PIASy and PIASy mutants were gifts from K. Shuai (University of California, Los Angeles, CA) and R. Grosschedl (University of Munich, Munich, Germany). Mammalian expression vectors for STAT1, PR-A, PR-B, and PR mutants have been previously described (13, 37). The amino-terminally His-tagged PR-A construct was created by PCR amplification of the PR-A coding region from pSG5/PR-B incorporating Acc65I and XbaI sites in the sense and antisense primers, respectively. The PCR product was digested with Acc65I/XbaI and inserted into the Acc65I/XbaI sites of pcDNA3.1/V5-His vector (Invitrogen Life Technologies). The V5-tagged mPR{alpha} expression vector was a gift from B. Gellersen (University of Hamburg, Hamburg, Germany).

Undifferentiated and decidualizing primary ESC cultures were transiently transfected in 24-well plates using calcium phosphate precipitation in medium supplemented with 2% DCC-FBS as described previously (13, 37). Decidualizing cells were pretreated with 0.5 mM 8-bromo-cAMP and 10–6 M MPA for 48 h before transfection, and this differentiation stimulus was maintained until cells were harvested. Promoter-reporter constructs and expression constructs were transfected at concentrations of 400 ng/well and 100 ng/well, respectively, unless indicated otherwise in figure legends. A ß-galactosidase control expression vector was cotransfected to control for transfection efficiency. Transfections were performed in triplicate and repeated at least three times. Representative experiments are shown (means ± SD).

GST Pull-Down Assays
GST pull-down assays were performed as described previously (38). 35S-labeled proteins were prepared by the in vitro transcription-translation method, using the TNT T7 Coupled Reticulocyte Lysate System (Promega, Madison, WI) following the supplier’s protocol. The presence of 35S-methionine (>1000 Ci/mmol, Amersham Pharmacia Biotech) in the incubation mixture was used to produce labeled PIASy and FoxO1a proteins from pSG5/PIASy and pcDNA3.1/FoxO1a, respectively.


   ACKNOWLEDGMENTS
 
We thank L. Naumovski (Stanford University, Stanford, CA) for the anti-Nmi antibody; K. Ozato (National Institutes of Health, Bethesda, MD) for the GAS-wt/luc and GAS-mt/luc reporter constructs; K. Shuai (University of California, Los Angeles, CA) for the PIASy and PIASy-AA expression vectors; and R. Grosschedl (University of Munich, Munich, Germany) for T7-PIASy and PIASy-RING plasmids. We are indebted to B. Gellersen (University of Hamburg, Hamburg, Germany) for the V5-mPR{alpha} plasmid, advice, and critical discussion.


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

Abbreviations: AR, Androgen receptor; DBD, DNA binding domain; DCC-FBS, dextran-coated charcoal-treated fetal bovine serum; DHT, dihydrotestosterone; E2, estradiol; ER, estrogen receptor; ESC, endometrial stromal cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GAS, IFN{gamma} activation site; GR, glucocorticoid receptor; GST, glutathione-S-transferase; HDAC, histone deacetylase; IFN-{gamma} interferon-{gamma}; IRF1, IFN response factor 1; JAK, Janus kinase; MPA, medroxyprogesterone acetate; mt, mutant; Ni-NTA, nickel nitrilotriacetic acid; Nmi, N-Myc interactor; NLS, nuclear localization signal; NP-40, Nonidet P-40; PR, progesterone receptor; PIAS, protein inhibitor of activated STAT; PR, progesterone receptor; PRE, progesterone response element; RTQ-PCR, real-time quantitative PCR; SDS, sodium dodecyl sulfate; STAT, signal transducer and activator of transcription; SUMO, small ubiquitin-like modifier; TSA, trichostatin A; uNK cells, uterine natural killer cells; wt, wild-type.

Received for publication December 3, 2003. Accepted for publication May 10, 2004.


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