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Molecular Endocrinology, doi:10.1210/me.2006-0539
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Molecular Endocrinology 21 (11): 2725-2737
Copyright © 2007 by The Endocrine Society

MUC1 Expression Is Repressed by Protein Inhibitor of Activated Signal Transducer and Activator of Transcription-y

Melissa Jo Brayman, Neeraja Dharmaraj, Errin Lagow and Daniel D. Carson

Department of Reproductive Medicine (M.J.B.), University of California, San Diego, School of Medicine, La Jolla, California 92093; Department of Biological Sciences (N.D., D.D.C.), University of Delaware, Newark, Delaware 19716; and Ordway Research Institute (E.L.), Cancer Center, Albany, New York 12208

Address all correspondence and requests for reprints to: Daniel D. Carson, Ph.D., Department of Biological Sciences, University of Delaware, 118C Wolf Hall, Newark, Delaware 19713. E-mail: dcarson{at}udel.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mucin 1 (MUC1) is a transmembrane glycoprotein that modulates the interaction between the embryo and the uterine epithelial cell surface. MUC1 also is a tumor marker and has been implicated in the protection of cancer cells from immune cell attack as well as in cell signaling in some tumors. We and others have shown that MUC1 expression is activated by progesterone (P), TNF-{alpha}, and interferon-{gamma} (IFN-{gamma}). Here we demonstrate that MUC1 expression is down-regulated by overexpression of members of the protein inhibitor of activated signal transducer and activator of transcription (PIAS) family, PIAS1, PIAS3, PIASx{alpha}, PIASxß, and PIASy, in human uterine epithelial cell lines HES and HEC-1A and in a breast cancer cell line, T47D. Treatments with P, TNF-{alpha}, and IFN-{gamma} were unable to overcome the repression by PIASy. PIASy repression of basal, P-, and TNF-{alpha}-stimulated MUC1 promoter activity was not dependent on the PIASy sumoylation domain. In contrast, PIASy suppression of IFN-{gamma}-activated MUC1 promoter activity was dependent on the PIASy sumoylation domain. PIASy and P receptor B were localized to the nucleus upon P treatment, and small interfering RNA knockdown of PIASy resulted in an increase in P-mediated stimulation of MUC1 protein expression. Overexpression of PIASy did not affect P receptor B binding to the MUC1 promoter but surprisingly led to a loss of nuclear receptor corepressor (NCoR), which was recruited to the promoter in response to P. Collectively, these data indicate that PIASy may be a useful target for down-regulation of MUC1 expression in various contexts.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MUCIN 1 (MUC1) IS a transmembrane glycoprotein that is expressed in a variety of epithelial cell types, including those of the female reproductive tract, mammary gland, lung, kidney, stomach, gall bladder, and pancreas (1). MUC1 is involved in the modulation of cell adhesion, lubrication and hydration of epithelial surfaces, and protection from infection (2, 3). Cells expressing a high level of MUC1 at the cell surface are refractory to blastocyst attachment, whereas removing MUC1 from the cell surface allows attachment (4, 5, 6). In addition, MUC1 is overexpressed and aberrantly glycosylated by a variety of tumors (1, 7, 8) and has been shown to protect cancer cells from immune cell attack (9, 10, 11).

We and others have shown that human MUC1 expression is regulated by progesterone (P) (12) and proinflammatory cytokines (13, 14, 15) in uterine and breast cancer cell lines. Human uterine MUC1 expression is up-regulated during the receptive period, and a localized loss of MUC1 at implantation sites is required for embryo implantation to occur (16, 17). The mechanism of local MUC1 loss is not known, although evidence suggests the involvement of locally activated sheddases (18, 19), a change in MUC1 gene expression (5, 12), or both. Identification of factors that reduce or down-regulate MUC1 expression would be beneficial for treatment of infertility, including in vitro fertilization, as well as cancer treatments; however, factors that reduce MUC1 expression have not been identified to date.

Members of the protein inhibitor of activated signal transducer and activator of transcription (STAT) (PIAS) family interact with a number of transcription factors to regulate their activity (20, 21). The family members share 40% homology and function as E3-type small ubiquitin-like modifier (SUMO) ligases, transcriptional coregulators, and DNA-interacting proteins (20, 22). Although PIAS proteins originally were identified as STAT-interacting factors by yeast 2-hybrid screening (21, 23), they also interact with nuclear receptors and other transcription factors, transcriptional cofactors, tumor suppressors, and other proteins (20, 24). PIAS proteins can activate or repress nuclear receptor activity, depending on the receptor, cell, and promoter type (25). In addition, members of the PIAS family may promote cross talk between distinct signaling pathways that are regulated by the same PIAS protein. For example, PIASy repression of STAT1 in endometrial stromal cells is dependent on liganded P receptor (PR) and vice versa (26).

MUC1 expression is maximal in human uterine luminal epithelium during the receptive period, yet MUC1 protein expression must be lost at implantation sites in order for implantation to occur. A number of transcription factors have been shown to stimulate MUC1 expression in uterine epithelium (UE) in response to a variety of signals (27). Several of the factors that have been shown to stimulate MUC1 expression, including STAT1, STAT3, nuclear factor-{kappa}B (NF{kappa}B), and PR, also are known to interact with various PIAS proteins (20, 24). Here we report that PIAS family members repress basal MUC1 transcriptional activity in human uterine epithelial cell lines HEC-1A and HES and in a breast cancer cell line, T47D. Stimulation of MUC1 expression by P, TNF-{alpha}, and interferon-{gamma} (IFN-{gamma}) also is repressed by PIASy. We report that PIASy repression of MUC1 promoter activity is independent of the PIASy sumoylation domain. We also observed that PIASy and PR interact in a P-dependent manner. This interaction does not affect binding of PR to the MUC1 proximal promoter region, although the recruitment of cofactors is altered. Collectively, these studies indicate that PIASy and certain other PIAS family members can attenuate MUC1 expression in response to various physiologically relevant stimulators.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PIAS Family Members Are Expressed by Uterine Epithelial Cells, Cell Lines, and Endometrium
Little is known about PIAS family member expression in uterine cell lines or endometrial tissues during the menstrual cycle. Therefore, we analyzed total RNA from HEC-1A and HES human uterine epithelial cell lines, the T47D breast cancer cell line, staged human endometrial biopsies, and isolated murine UE by RT-PCR (Fig. 1Go). Whole mouse embryo RNA was used as a control. PIAS1, PIAS3, PIASx{alpha}, and PIASy were detected by all three cell lines tested, although PIAS1 and PIAS3 were detected at low levels in HEC-1A, and PIAS3 was detected at low levels in HES. PIASxß was expressed in HES and T47D. All five PIAS family members examined were detected in early, mid, and late luteal phase human endometrium. PIAS3, PIASx, and PIASy were expressed by mouse UE; PIASx and PIASy were detected in whole mouse embryo, with a low level of PIAS1 detected. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a load control. Consequently, we concluded that PIAS family members were expressed appropriately to play a role in modulation of MUC1 expression.


Figure 1
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  		Fig. 1. RT-PCR Analysis of PIAS Family Member Expression in Various Cell Types and Cell Lines

Total RNA from HEC-1A, HES, or T47D cell lines, staged human endometrial biopsies (EL, early luteal; LL, late luteal; ML, mid-luteal), mouse UE cells, and whole-mouse embryo was reverse-transcribed, and the resulting cDNA was amplified using primers to the indicated PIAS family members or GAPDH mRNAs. GAPDH was used as a load control.

 
Overexpression of PIAS Family Members in HEC-1A Cells Results in Decreased Basal and P-Stimulated MUC1 Promoter Activity
To determine whether overexpression of PIAS family members had an effect on MUC1 promoter activity in HEC-1A cells, cells were transiently cotransfected with the 1.4MUC reporter plasmid, with or without Flag-PIAS1, Flag-PIAS3, Flag-PIASx{alpha}, Flag-PIASxß, or Flag-PIASy. Overexpression of any PIAS protein resulted in the repression of basal MUC1 promoter activity (Fig. 2AGo). When PRB was coexpressed with the PIAS family members, PIAS1 and PIASy repressed P-mediated activity to the greatest extent, whereas PIAS3 displayed a partial inhibition. PIASxß did not significantly repress PRB-mediated activation, and PIASx{alpha} had a marginal effect.


Figure 2
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  		Fig. 2. PIAS Proteins Inhibit Basal and P-Stimulated MUC1 Promoter Activity

A, HEC-1A cells were transiently transfected with the 1.4MUC reporter together with hPRB and vector (control), Flag-PIAS1, Flag-PIAS3, Flag-PIASx{alpha}, Flag-PIASxß, or Flag-PIASy in the absence or presence of P treatment (400 nM); B, HEC-1A cells were transiently transfected with the 604/487MUC reporter together with hPRB and vector (control) or the indicated Flag-PIAS expression plasmid in the absence or presence of P treatment; C, HEC-1A cells were transiently transfected with the pGL3-SV40 reporter and vector (control) or the indicated Flag-PIAS expression plasmid; D, HEC-1A cells were transiently transfected with a PRE-TK reporter, hPRB, and vector (control) or the indicated Flag-PIAS expression plasmid in the absence or presence of P treatment. White bars denote vehicle; black bars denote P treatment. Bars represent luciferase activity relative to vector control; numbers above each bar represent fold response to P treatment, relative to vehicle-treated. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. no PIAS.

 
We previously have shown that PRB regulates MUC1 promoter activity through the region between 564 and 487 bp upstream of the start site of transcription (12). To determine whether this region contains the cis-elements required for PIAS repression of PRB-mediated activation, a heterologous promoter containing the region between 604 and 487 bp upstream of the MUC1 transcriptional start site (604/487MUC) was used. This fragment contains the P-responsive region as well as binding sites for NF{kappa}B, STAT, and Sp1 (12, 13, 14). The 604/487MUC construct was transiently transfected into HEC-1A cells, along with hPRB, with or without Flag-PIAS1, Flag-PIAS3, or Flag-PIASy. Upon treatment with P, a repression of P-stimulated promoter activity by all three PIAS proteins was observed (Fig. 2BGo), indicating that this region is sufficient for PIAS antagonism of PRB activation. The STAT binding site located in this region was not involved in this repression, because mutation of the site had no effect on PIAS antagonism (data not shown). In addition, PIAS1 and PIASy repressed P-stimulated activity from a shorter MUC1 promoter fragment (564/500MUC) that contains the P-responsive elements but not the NF{kappa}B, STAT, or Sp1 binding sites (data not shown). Thus, PIAS repression of P responsiveness does not require elements outside of the P-responsive region of the MUC1 promoter.

To determine whether the PIAS effect on basal promoter activity was specific for the MUC1 promoter, HEC-1A cells were transiently transfected with a plasmid containing the SV40 promoter and enhancer upstream of the firefly luciferase gene (pGL3-SV40), with or without each PIAS expression plasmid. Only PIAS1 significantly repressed SV40 promoter activity (Fig. 2CGo), suggesting that repression of MUC1 by the other PIAS family members was not due to a general repression of the basal transcriptional machinery.

To determine whether the PIAS effect on P-stimulated MUC1 promoter activity was due to a global effect on PR action, HEC-1A cells were transiently transfected with hPRB and a reporter plasmid containing a consensus P response element (PRE) upstream of the thymidine kinase promoter and a luciferase gene (PRE-TK), with or without each PIAS expression plasmid, and then treated with P for 24 h. A similar repression of PRE-TK promoter activity by the PIAS proteins was seen (Fig. 2DGo) compared with repression of 1.4MUC promoter activity, suggesting that the PIAS effects on PRB-mediated stimulation were not specific for the MUC1 promoter, but likely involved other PR-regulated genes.

Overexpression of proteins can result in nonspecific interactions. To determine whether this was the case in our system, increasing amounts of Flag-PIAS1, Flag-PIAS3, or Flag-PIASy expression plasmids were transiently transfected into HEC-1A cells. Each of the three PIAS family members repressed PRB-mediated MUC1 promoter activity in a PIAS dose-dependent manner (Fig. 3Go). PIAS1, PIAS3, and PIASy repressed both basal and P-stimulated activity from the MUC1 promoter, regardless of the amount of plasmid used. Based on the data presented above, PIASy was the most effective and specific PIAS protein at repression of MUC1; therefore, the effect of PIASy on MUC1 expression was investigated further.


Figure 3
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  		Fig. 3. Dose Response of PIAS1, PIAS3, and PIASy Action on MUC1 Promoter Activity

HEC-1A cells were transfected with hPRB and 1.4MUC together with increasing amounts of Flag-PIAS1, Flag-PIAS3, or Flag-PIASy (0, 0.2, 1, or 2 µg) in the absence (white bars) or presence (black bars) of P treatment (400 nM). Bars represent luciferase activity relative to vehicle-treated vector control (–); numbers above each bar represent fold response to P treatment, relative to vehicle-treated. ***, P < 0.001 vs. no PIAS.

 
PIASy Represses Liganded PRA- and PRB-Mediated Activation of the MUC1 Promoter
PIAS proteins can activate or repress nuclear receptor activity, depending on the cellular context (25). In addition, PRA and PRB often are differentially regulated by cofactors (28). Thus, the effect of PIASy overexpression on PRA-mediated activity and transrepression of PRB was evaluated. HEC-1A cells were transiently transfected with 1.4MUC and hPRA and/or hPRB, with or without Flag-PIASy. As expected, overexpression of PIASy repressed P stimulation of MUC1 promoter activity mediated by PRB (Fig. 4Go). As shown previously (12), PRA only modestly stimulated MUC1 expression and antagonized PRB action, in this regard. Nonetheless, PIASy reduced the modest stimulation of MUC1 promoter activity by PRA and also repressed MUC1 promoter activity in the presence of both PR isoforms. These observations demonstrated that PIASy did not differentially regulate the transcriptional activities of the two PR isoforms and that PIASy did not interfere with PRA transrepression of PRB activity.


Figure 4
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  		Fig. 4. PIASy Does Not Affect PRA Transrepression of PRB

HEC-1A cells were transiently transfected with 1.4MUC together with hPRA, hPRB, and/or Flag-PIASy in the absence (white bars) or presence (black bars) of P treatment (400 nM), as indicated. Bars represent luciferase activity relative to vector control; numbers above each bar represent fold response to treatment, relative to vehicle-treated. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. indicated treatment.

 
PIASy-Mediated Antagonism of MUC1 Promoter Activity in Other Cell Lines and in Response to Other Agonists
We next sought to determine whether PIASy actions with regard to MUC1 expression were unique to the HEC-1A cell line or might be a general action. The HES cell line, a PR-negative uterine epithelial cell line, was transiently transfected with 1.4MUC and hPRB, with or without Flag-PIASy, and treated with P for 24 h (Fig. 5AGo). T47D, a PRA- and PRB-positive breast cancer cell line, were transiently transfected with 1.4MUC, with or without Flag-PIASy, and treated with a progestin, R5020, for 24 h (Fig. 5BGo). PIASy antagonized both basal and P-stimulated MUC1 promoter activity in these cell lines, demonstrating that PIASy regulation of the MUC1 promoter was not cell-type specific and that overexpression of PIASy repressed endogenous PRB activity.


Figure 5
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  		Fig. 5. PIASy Inhibits Basal, P-Stimulated, and Cytokine-Stimulated MUC1 Promoter Activity in HES and T47D Cell Lines

A, HES cell line was transiently transfected with 1.4MUC and hPRB and vector (control) or Flag-PIASy in the absence (white bars) or presence (black bars) of P treatment (400 nM); B, same as A, except T47D cell line was used and cells were treated with vehicle (white bars) or 1 nM R5020 (black bars); C, HES cells were transfected with 1.4MUC with or without Flag-PIASy and treated with vehicle (white bars), 25 ng/ml TNF-{alpha} (hatched bars), 250 IU IFN-{gamma} (cross-hatched bars), or 25 ng/ml TNF-{alpha} plus 250 IU IFN-{gamma} (black bars); D, same as C, except the T47D cell line was used. Bars represent luciferase activity relative to vector control; numbers above each bar represent fold response to treatment, relative to vehicle-treated. **, P < 0.01; ***, P < 0.001 vs. no PIAS.

 
We and others have shown that TNF-{alpha} and IFN-{gamma} activate MUC1 promoter activity in a synergistic manner via NF{kappa}B and STAT1 binding, respectively (13, 14, 15). PIASy has been shown to interact with STAT1 (20, 24) and can modulate the NF{kappa}B pathway (29, 30). PIASy repressed TNF-{alpha} and IFN-{gamma} stimulation of MUC1 promoter activity, as well as the synergistic response, in both HES (Fig. 5CGo) and T47D (Fig. 5DGo) cell lines. Thus, PIASy inhibited STAT1- and NF{kappa}B-mediated activation of MUC1 promoter activity. Collectively, these data indicated that overexpression of PIASy repressed MUC1 promoter activity regardless of treatment or cell type. A lower ratio of PIAS plasmid to 1.4MUC was transfected into the HES cells that were treated with cytokines, resulting in a less dramatic repression of promoter activity. Transfection of HES cells with equal amounts of 1.4MUC and empty vector (or Flag-PIASy), followed by treatment with TNF-{alpha} and/or IFN-{gamma}, resulted in increased cell death and loss of cytokine stimulation of MUC1 promoter activity (data not shown).

PIASy-Mediated Repression of Basal and P-Stimulated MUC1 Promoter Activity Is Improved by the PIASy Sumoylation Domain
PIAS proteins can function as E3-type SUMO ligases (31), although sumoylation of nuclear receptors is not always required for PIAS corepressor function (32, 33). On the other hand, SUMO-1 modification of the PR N terminus is required for PR autoinhibition and transrepression (34), suggesting that PRB sumoylation by PIASy may be important in the repression of P-stimulated MUC1 promoter activity. To investigate this possibility, the ability of PIASy with a mutation (W363A) in the sumoylation domain (Flag-mutPIASy) to repress basal and P-stimulated MUC1 promoter activity was investigated. HEC-1A cells were transiently transfected with 1.4MUC, with a vector control (CMV5-Flag), Flag-PIASy, or Flag-mutPIASy. Overexpression of PIASy repressed basal 1.4MUC activity by more than 30% (Fig. 6AGo). Slightly less repression of basal MUC1 promoter activity was observed when mutPIASy was cotransfected (25%), indicating that sumoylation enhanced, but was not required for, PIASy inhibition of basal MUC1 promoter activity.


Figure 6
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  		Fig. 6. The PIASy Sumoylation Domain Enhances PIASy-Mediated Inhibition of MUC1 Promoter Activity

A, HEC-1A cell line was transiently transfected with 1.4MUC and vector (control), Flag-PIASy, or a PIASy expression plasmid with a mutation in the sumoylation domain (Flag-mutPIASy). Data are expressed as luciferase activity relative to control. B, HEC-1A cells were transfected with 1.4MUC and hPRB together with various amounts of Flag-PIASy or Flag-mutPIASy (0, 0.02, 0.1, 0.2, or 2 µg) in the absence or presence of P treatment (400 nM). Data are expressed as the fold activation in response to treatment, relative to vehicle-treated. C, T47D cells were transfected with 1.4MUC and vector (control), Flag-PIASy, or Flag-mutPIASy in the absence or presence of 25 ng/ml TNF-{alpha} (hatched bars), 250 IU IFN-{gamma} (cross-hatched bars), or 25 ng/ml TNF-{alpha} plus 250 IU IFN-{gamma} (black bars). Data are expressed as the fold activation in response to treatment, relative to vehicle-treated. **, P < 0.01; ***, P < 0.001 vs. no PIAS; ^, P < 0.05; ^^, P < 0.01; ^^^, P < 0.001 vs. PIASy.

 
It is possible that overexpression of PIASy allows repression of transcription factor activity in the absence of sumoylation, even if sumoylation is required under physiological conditions. To assess this possibility, increasing amounts of Flag-PIASy and Flag-mutPIASy were cotransfected into cells with 1.4MUC and hPRB. At all plasmid concentrations used, mutPIASy was significantly less capable of repressing P-stimulated MUC1 promoter activity than was wild-type PIASy (Fig. 6BGo); however, even at an amount 1/100 less than was used previously, mutPIASy significantly inhibited PRB-mediated activation. A SUMO-specific protease, sentrin/SUMO-specific protease-1 (SENP-1), has been shown to reverse sumoylation of several proteins (35, 36). Interestingly, coexpression of SENP-1 has no effect on PIASy repression of basal or PRB-mediated activation (data not shown). The PIASy sumoylation domain also was not required for antagonism of TNF-{alpha} stimulation in T47D cells, but it was necessary for the antagonism of IFN-{gamma} stimulation (Fig. 6CGo), although a slight reduction in responsiveness to IFN-{gamma} was observed when the site was mutated. We concluded that the SUMO ligase activity of PIASy 1) was required for repression of STAT1 activity, 2) enhanced, but was not required for, PIASy function as a PRB corepressor and, 3) was not required for repression of NF{kappa}B activity.

Endogenous PIASy Represses P Responsiveness of MUC1 Expression
Small interfering RNA (siRNA) was used to knock down endogenous PIASy in HEC-1A cells that stably express PRB (PRB3). PIASy mRNA was reduced by between 28 and 59% upon transfection of the cells with siRNA specific for PIASy (Fig. 7AGo). Basal MUC1 protein expression was not affected by depletion of PIASy (Fig. 7BGo); however, MUC1 expression was stimulated to a much greater extent in response to P treatment when endogenous PIASy was knocked down (Fig. 7Go, B and C). These findings indicate that endogenous PIASy attenuates P-activated MUC1 protein expression, consistent with the actions on MUC1 promoter activity.


Figure 7
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  		Fig. 7. PIASy Knockdown by siRNA Increases P Responsiveness of MUC1 Expression

PRB3 cells were transfected with a control siRNA or PIASy siRNA and then treated with or without 400 nM P for 24 h before assay. A, PIASy knockdown was evaluated by RT-PCR; B, MUC1 expression was evaluated by Western blot with an antibody to the MUC1 tandem repeats, and GAPDH protein was used as a load control; C, densometric analysis of MUC1 protein. Data are expressed as fold P response (relative to vehicle-treated control) relative to GAPDH. White bars denote vehicle, black bars denote P treatment. ***, P < 0.001 vs. control P response.

 
PIASy and PRB Localize to the Nucleus in P-Treated Cells
Members of the PIAS family can repress transcription factor activity by a number of pathways, including recruitment of corepressors, altering the subnuclear localization of the transcription factor, and recruitment to nuclear bodies (20, 37, 38). Such repression appears to require direct interaction between the transcription factor and PIAS protein. To examine the subcellular localization of PIASy and PRB, PRB3 cells were treated with or without P for 1 h, followed by immunofluorescence analysis with antibodies specific for PIASy or nuclear PR. Some PR was seen in the nuclei of vehicle-treated cells, and the nuclear localization of PR was increased by P treatment (Fig. 8Go, A and E). Staining for PIASy was negligible in untreated cells (Fig. 8BGo). This likely was due to the masking of epitopes in the cytoplasm, because a second set of antibodies showed diffuse PIASy staining throughout the cell before treatment (data not shown). PIASy was recruited to the nucleus upon treatment with P (Fig. 8FGo). Thus, both PRB and PIASy were localized in the nuclei of P-treated PRB3 cells, with a few distinct foci observed (Fig. 8HGo). Fewer foci were observed when a second set of antibodies was used (data not shown), possibly due to a difference in the epitopes recognized by these antibodies.


Figure 8
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  		Fig. 8. PIASy and PRB Colocalize in the Nucleus of P-Treated Cells

HEC-1A cells that had been stably transfected with hPRB and treated with or without P for 1 h were subjected to immunofluorescence assays with antibodies specific for PR (A and E) and PIASy (B and F), Draq5 stain for DNA (C and G), or the merged images (D and H). Bar, 5 µm; magnification is the same for all panels.

 
Interaction of PIASy and PRB in HEC-1A Cells
To determine whether PIASy and PRB physically interact when coexpressed in PRB3 cells, cells were transfected with or without Flag-PIASy, in the presence or absence of P treatment for 1 h. Cell lysates were immunoprecipitated with an anti-Flag antibody and immunoblotted with an antibody specific for PR. A band corresponding to PRB was observed only in P-treated cells that had been transfected with Flag-PIASy (Fig. 9AGo), indicating that PIASy and PRB interacted in vivo only in the presence of ligand. However, these data do not rule out the possibility that PIASy and PRB interact via another protein and do not directly bind one another. No PR was detected when immunoprecipitation was performed with normal mouse serum (data not shown).


Figure 9
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  		Fig. 9. PIASy Interacts with PR and Alters Recruitment of Cofactors to the MUC1 Promoter

HEC-1A cells were transiently transfected with vector or Flag-PIASy in the presence or absence of P treatment. A, Coimmunoprecipitation was performed on equivalent amounts of cellular protein with a Flag monoclonal antibody and then analyzed by immunoblotting with a PR monoclonal antibody; B, ChIP assays were performed on equivalent amounts of nuclear protein with normal rabbit serum or an antibody specific for PR, Flag, SRC-1, SRC-3, SMRT, or NCoR, as indicated. PCR was performed with primers to the P-responsive region of the MUC1 promoter. Input lanes represent 1/60 total input chromatin.

 
Overexpression of PIASy Alters Cofactor, But Not PRB, Recruitment to the P-Responsive Region of the MUC1 Proximal Promoter
PIASy is able to interact with both transcription factors and their coregulatory proteins. PIASy-interacting cofactors include the members of the steroid receptor coactivator (SRC) family, and the corepressors, silencing mediator of retinoid and thyroid receptors (SMRT) and nuclear receptor corepressor (NCoR) (20, 24, 40). To determine whether overexpression of PIASy disrupts PRB and/or nuclear receptor coregulator interaction with the MUC1 promoter, PRB3 cells were transiently transfected with or without Flag-PIASy and treated with P for 1 h before chromatin isolation and precipitation with antibodies specific for PR, Flag, SRC-1, SRC-3, SMRT, or NCoR. We have shown previously that PRB interacts with the P-responsive region (618–500 bp upstream of the transcriptional start site), but not a control region (–163 to –23), of the MUC1 promoter (12). PRB was recruited to the P-responsive region of the MUC1 promoter upon P treatment, regardless of PIASy overexpression (Fig. 9BGo). Conversely, Flag-PIASy was not recruited to the MUC1 promoter. Surprisingly, SMRT and NCoR were recruited to the MUC1 promoter, whereas SRC-3 association with the promoter decreased, upon P treatment. SRC-1 was constitutively associated with the promoter region, regardless of treatment. NCoR association with the P-responsive region of the MUC1 promoter was completely disrupted by overexpression of PIASy, whereas SRC-3 association increased. SRC-1 and SMRT were unaffected by PIASy overexpression. These data were surprising, because they indicated that increased corepressor binding to the promoter was associated with transcriptional activation by PR, and the loss of NCoR association was associated with the loss of this activation. No association with the control promoter region was observed, regardless of the treatment or protein examined (data not shown).

PIASy has been shown to repress nuclear receptor activity by recruiting histone deacetylases (HDACs) to the promoters of target genes (33, 39); however, treatment with a HDAC inhibitor, trichostatin A, had no effect on PIASy repression of PRB-activated or basal MUC1 promoter activity (data not shown). Taken together, these studies indicate that PIASy physically interacts with PRB and inhibits its ability to activate transcription even when bound to the MUC1 promoter, perhaps by sequestration of cofactors required for MUC1 transcriptional activation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The PIAS proteins originally were identified as STAT inhibitors, and they also have been shown to regulate nuclear receptor activity in a variety of contexts. Human MUC1 gene expression is regulated by PRs (12), NF{kappa}B, and STATs (13, 14), making it a potential target of PIAS regulation as well. This study showed that all five mammalian PIAS family members, PIAS1, PIAS3, PIASx{alpha}, PIASxß, and PIASy, were expressed by human uterine cells and uterine epithelial cell lines as well as by the T47D breast cancer cell line and mouse UE. Whether PIAS protein expression or activity is regulated through the menstrual cycle and pregnancy remains to be determined.

Overexpression of each PIAS protein tested repressed both basal and PRB-stimulated MUC1 promoter activity in HEC-1A cells, although PIASx{alpha} and PIASxß had only modest activity in this regard. This repression did not require cis elements outside of the –604 to –487-bp region of the MUC1 promoter, a region that contains several transcription factor binding sites, including those for Sp1, NF{kappa}B, STAT, and PR. The STAT binding site is not involved in the repression, because mutation of the STAT binding site (at –503/–495) did not affect PIASy antagonism of MUC1 promoter activity (data not shown). The PIAS-mediated antagonism of basal MUC1 promoter activity appeared to be specific for the MUC1 promoter, because most PIAS proteins failed to repress the activity of the SV40 promoter and enhancer. On the other hand, PIAS1, PIAS3, PIASxß, and PIASy all were capable of repressing the P responsiveness of a consensus PRE, suggesting that this repression is not restricted to the MUC1 promoter and may reflect a more general control of PR action.

PIASy is highly expressed in the human uterine cell lines HEC-1A and HES as well as in human and murine uterine cells in vivo. PIASy repressed MUC1 basal and P-stimulated promoter to the greatest extent and with the highest specificity of the PIAS proteins tested. Therefore, PIASy was used to further evaluate PIAS regulation of MUC1 promoter activity. Overexpression of PIASy repressed both PRA- and PRB-mediated activity and had no effect on PRA transrepression of PRB activity. PIASy may regulate both of the PR isoforms by the same mechanism. In addition, PIASy repressed basal and PRB-, TNF-{alpha}-, and IFN-{gamma}-stimulated MUC1 promoter activity in the HES and T47D cell lines. Although the repression of cytokine-stimulated activity was not as robust in the HES as in the T47D cell line, this likely was due to the lower amount of Flag-PIASy used in relation to 1.4MUC. These data indicate that the effect of PIASy on MUC1 expression is not cell line dependent and cannot be overcome by any of the treatment conditions examined. Endogenous PIASy also repressed P-stimulated MUC1 protein expression. However, siRNA knockdown of PIASy did not affect basal MUC1 expression, perhaps because some PIASy was still present in these cells or because other PIAS proteins compensated for the loss of PIASy.

Antagonism of transcriptional activation by PIAS proteins may be due to a variety of actions, including inhibition of transcription factor binding to the promoter, recruitment of HDACs, recruitment of corepressors, and/or interference with coactivator binding. These actions can be either dependent on or independent of SUMO ligase activity. We investigated several of these possibilities with regard to MUC1 expression. PIASy repression of basal, P-stimulated, and TNF-{alpha}-stimulated MUC1 promoter activity is enhanced by, but does not require, the PIASy sumoylation domain. PIASy can inhibit transcription factor activity in a sumoylation-independent manner (22, 33, 38), although forced overexpression of PIASy may overcome the need for sumoylation in our system, or other SUMO ligases may be involved. The latter explanation is unlikely, because overexpression of SENP-1, a SUMO-specific protease, did not reverse PIASy repression of MUC1 expression (data not shown). In contrast, PIASy antagonism of IFN-{gamma}-stimulated MUC1 promoter activity did require the PIASy sumoylation domain. Previous studies indicated that the PIASy SUMO ligase domain was not required for PIASy inhibition of IFN-{gamma} stimulation in decidualized uterine stromal cells (26). Thus, these actions may be due to differences in cell type or promoter context. IFN-{gamma} has been shown to activate MUC1 expression via STAT1, a PIASy-interacting protein (21). Mutation of the STAT binding site did not affect PIASy antagonism of PRB activity, indicating that STAT proteins are not involved in PIASy repression of basal or P-stimulated MUC1 promoter activity. It also is unlikely that PIASy interaction with a member of the basal transcriptional machinery (e.g. RNA polymerase II, transcription factors II, and TATA-binding protein) is involved in its repression of MUC1 expression, because the reporter construct carrying the SV40 promoter and enhancer was not affected by PIASy overexpression.

PIAS proteins have been shown to alter nuclear receptor localization in the nucleus, by recruitment to nuclear bodies (22, 37) or other subnuclear compartments (38). Although PRB and PIASy are both recruited to the nucleus and interact in a P-dependent manner, it remains to be determined whether this interaction is direct or requires an intermediary, such as a coregulatory protein. Nonetheless, this interaction does not affect PRB recruitment to the P-responsive region of the MUC1 promoter. PIASy antagonism of PRB activity, therefore, is not due to sequestration of PRB from the promoter. Unexpectedly, Flag-PIASy was not recruited to the P-responsive region of the MUC1 promoter, suggesting that the interaction between PRB and PIASy was not sufficient to explain the loss of PR-mediated activation. Alternatively, the amount of Flag-PIASy that was transfected into the cells and associated with the promoter was too low to be detected using these methods. Another explanation may be that PIASy binds to a different region of the MUC1 promoter, although this is unlikely considering that the P-responsive region was sufficient for PIASy-mediated repression in transient transfection assays.

The associations of NCoR and SMRT with the MUC1 promoter increased upon P treatment, and NCoR association was lost upon overexpression of PIASy, perhaps due to an increase in NCoR-PIASy interactions. The increase in corepressor association with the activated promoter is puzzling and may represent a novel activation function of these proteins, as has been shown recently for SMRT (41). In addition, NCoR has been shown to augment transcriptional activation by unliganded thyroid hormone receptor (42, 43, 44). Alternatively, the recruitment of corepressors to the activated promoter may represent complex regulation of MUC1 transcription by oscillation of cofactor binding over time (45). Another possibility is that other coregulatory factors may be more important for MUC1 transcriptional activation in response to P treatment, and PIASy could disrupt the interaction of these other cofactors with the MUC1 promoter. It is unclear from our studies whether the cofactors examined are recruited to the MUC1 promoter by direct interaction with PR or through interaction with another transcription factor present the promoter region. Our studies with the general HDAC inhibitor trichostatin A indicate that HDAC activity also is not required for PIASy-mediated repression (data not shown). The exact mechanism of PIASy repression of MUC1 promoter activity remains to be determined, and the involvement of cofactors in the regulation of MUC1 expression presents an intriguing direction for future work.

MUC1 expression is stimulated by P, probably via PRB, in the human endometrium. To date, few factors or treatments have been shown to down-regulate MUC1 expression in uterine epithelial cells, yet MUC1 expression must be lost at implantation sites for embryo implantation to occur. This study showed that the PIAS protein family represents a potential source of MUC1-repressive factors in the uterus and that these factors can dramatically repress basal MUC1 promoter activity. This repression is not wholly overcome by treatment with P, TNF-{alpha}, or IFN-{gamma}, factors that have been shown to stimulate MUC1 expression. Study of the role of PIAS proteins in the localized loss of MUC1 expression at human implantation sites awaits the development of a good model for human implantation. Additionally, few reports have evaluated the regulation of PIAS expression and/or activation in any cell type. Future work should be done to address whether PIAS proteins are constitutively active or whether their expression and/or activity are regulated, e.g. by signaling pathways, the menstrual cycle, the cell cycle, etc. There is some evidence for steroid hormone regulation of PIAS protein expression; PIAS3 expression is stimulated in response to dihydroxytestosterone treatment in prostate cancer cells (46). Increased knowledge of PIAS protein expression and regulation in uterine epithelial cells will allow a better understanding of the role of PIAS family members in the regulation of MUC1 gene expression and whether PIAS proteins play a role in embryo implantation and uterine development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids and Cell Lines
The pGL3-SV40 (pGL3-control) and pRL-TK plasmids were purchased from Promega (Madison, WI). The 1.4MUC (a gift from Sandra Gendler, Mayo Clinic, Scottsdale, AZ) (13), PRE-TK (47), 604/487MUC and 564/500MUC (12), and hPRB (a gift from Dr. Pierre Chambon, College de France, Paris, France) (48) plasmids have been described previously. Flag-pCMV5, mouse Flag-PIAS1 (23), mouse Flag-PIAS3 (49), human Flag-PIASx{alpha}, human Flag-PIASxß, human Flag-PIASy, and Flag-mutPIASy (40, 50) were generous gifts of Dr. Ke Shuai (University of California, Los Angeles, CA).

The human endometrial carcinoma cell line HEC-1A was purchased from American Type Culture Collection (Manassas, VA). Cells were maintained in DMEM/F12 without phenol red (Invitrogen, Carlsbad, CA), mented with 10% (vol/vol) charcoal-stripped fetal bovine serum (FBS; HyClone, Logan, UT). HEC-1A cells stably transfected with hPRB (PRB3) were created and maintained as described previously (12). The human uterine epithelial cell line HES was kindly provided by Dr. Doug Kniss (Ohio State University, Columbus, OH) and was grown in DMEM (Invitrogen) mented with 5% (vol/vol) FBS (Invitrogen) and 1 mM sodium pyruvate (Sigma-Aldrich, St. Louis, MO). T47D human breast ductal carcinoma cells were obtained from American Type Culture Collection and maintained in RPMI 1640 medium (Invitrogen) mented with 10% (vol/vol) FBS.

RNA Isolation and RT-PCR
HEC-1A, HES, and T47D cells were plated in six-well plates and maintained as described until cells reached confluence. Total RNA was isolated using the RNeasy kit (QIAGEN, Valencia, CA) according to the manufacturer’s instructions and stored at –80 C. Total RNA from mouse embryo was purchased from Ambion (Austin, TX). Samples and total RNA from early, mid, and late luteal phase human endometrium were obtained and isolated as described (51). Total RNA from uterine epithelial cells was prepared from excised uteri of randomly cycling CF-1 mice as described previously (52).

Quantitation and estimation of purity were performed by measurement at UV wavelengths of 260 and 280 nm. One microgram of total RNA was reverse transcribed as described (12). PCR was performed using the GoTaq Green PCR kit (Promega) according to the manufacturer’s instructions. PCR primers used are given in Table 1Go; GAPDH primers were described previously (12). PCR was carried out under the following conditions: 1) 95 C for 2 min; 2) 30 cycles of 95 C for 30 sec, 59 C for 30 sec, and 72 C for 30 sec; and 3) 72 C for 5 min.


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  		Table 1. PIAS PCR Primers

 
Immunofluorescence Analysis
PRB3 cells (12) were plated in eight-well chambered cover glass slides (Nalgene Nunc International, Naperville, IL) coated with growth factor reduced Matrigel matrix (BD Biosciences, San Jose, CA) and incubated at 37 C for 16 h. Cells were changed to serum-free media for 24 h and then treated with 400 nM P (Sigma) or ethanol for 1 h before immunofluorescence analysis. Cells were fixed with 4% (vol/vol) formaldehyde in PBS (Electron Microscopy Sciences, Fort Washington, PA) for 10 min, followed by two washes with PBS and treatment with 0.2% (vol/vol) Triton X-100 for 5 min. Cells were washed twice with PBS and incubated in blocking solution [3% (wt/vol) BSA in PBS] for 1 h at room temperature. Incubation with mouse monoclonal anti-PR (PgR Ab-8; Lab Vision Corp., Fremont, CA) was performed at room temperature for 1 h, followed by washing and incubation with sheep antimouse IgG-fluorescein isothiocyanate (Jackson Immunoresearch, West Grove, PA) at a 1:200 dilution in PBS for 45 min at room temperature. Cells were washed and incubated with goat polyclonal anti-PIASy antibody [PIASy (P-18);, Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:100 dilution overnight at 4 C. After washes with blocking solution, donkey antigoat Alexa-Fluor 594 (Invitrogen Molecular Probes, Carlsbad, CA) was added at a 1:200 dilution in blocking solution along with nuclear counterstain (1:2000) Draq 5 (Biostatus Ltd., Leicestershire, UK) for 40 min at room temperature. Cells were washed with PBS and stored at 4 C until imaged on a Zeiss LSM 510 confocal microscope (Carl Zeiss Inc., Göttingen, Germany). Negative controls were performed by preincubation of PIASy antibody with specific PIASy peptide and with secondary antibodies alone for both ethanol- and P-treated cells (data not shown). Another set of antibodies to PIASy (IMG-290; Imgenex Corp., San Diego, CA) and PR (PgR Ab-2) were used with similar results (data not shown).

Transient Transfection and Reporter Assays
Transient transfections of HEC-1A, PRB3, and T47D cell lines were performed as described (12, 13). T47D cells were switched to RPMI 1640 medium containing 10% (vol/vol) charcoal-stripped FBS and 1 nM estrogen (Sigma) upon plating cells for transfection. Two micrograms of the indicated Flag-PIAS and/or SENP-1 vector were added per well, unless otherwise indicated. For transient transfections where varying amounts of PIAS plasmid were added per well, empty vector (pCMV5) was added to keep the total DNA per well constant. T47D cells respond more robustly to R5020 than to P (data not shown); thus, R5020 was used for experiments in this cell line. R5020 was purchased from Sigma.

HES cells were plated in six-well plates that had been coated with growth factor reduced Matrigel in DMEM mented with 10% (vol/vol) charcoal-stripped FBS and 1 mM sodium pyruvate. When cells reached 80–90% confluence, transient transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. 1.4MUC (250 ng) and either 250 ng Flag-PIASy and hPRB plasmids and 62.5 ng pRL-TK plasmid (for P treatment) or 75 ng Flag-PIASy and 31.25 ng pRL-TK plasmid (TNF-{alpha} and IFN-{gamma} treatment) were added per well. The pSG5 empty vector was used to keep total plasmid DNA added per well constant. Transfection medium was replaced 6 h after transfection with fresh medium containing 10% (vol/vol) charcoal-stripped FBS, and cells were allowed to recover overnight. TNF-{alpha} and/or IFN-{gamma} or vehicle [0.1% (wt/vol) BSA in PBS] were added in DMEM with 10% (vol/vol) charcoal-stripped FBS for 24 h. Cells were harvested and samples assayed as described (12).

For coimmunoprecipitation and chromatin immunoprecipitation (ChIP) assays, PRB3 cells were grown in 150-cm2 cell culture flasks until they were 80–90% confluent. Cells were transiently transfected with either 30 µg Flag-pCMV5 or 30 µg Flag-PIASy using 225 µl Lipofectamine 2000 (Invitrogen) in DMEM/F12 per flask. The cells were allowed to recover for 12 h in DMEM/F12 containing 1% (vol/vol) charcoal-stripped FBS before treatment with 0.001% (vol/vol) ethanol vehicle or 400 nM P for 1 h.

siRNA Knockdown
PRB3 cells were plated in 24-well plates and maintained as described until cells were 50–60% confluent, when serum-free DMEM/F12 was added for 24 h. Cells were transfected with 100 nM control (sc-44230; Santa Cruz Biotechnology) or PIASy (sc-40851) siRNA using 7 µl oligofectamine (Invitrogen) in 100 µl/well OptiMEM I (Invitrogen), and treatments were added 24 h after transfection. Cells were harvested using the RNeasy mini kit (QIAGEN) for RT-PCR analysis or in 100 µl sample extraction buffer [0.05 M Tris (pH 7.0), 8 M urea, 1.0% (wt/vol) SDS, 0.01% (vol/vol) phenylmethylsulfonyl-fluoride, and 1.0% (vol/vol) ß-mercaptoethanol] for Western blot analysis. RT-PCR was performed as described above; PCR was performed using the primers specific for PIASy and conditions described above, with the exception that 28 cycles were performed.

Coimmunoprecipitation Analysis
Cells were lysed in radioimmune precipitation assay buffer [50 mM Tris-Cl, (pH 7.8), 150 mM NaCl, 5 mM EDTA, 15 mM MgCl2, 0.5% (vol/vol) Nonidet P-40, 0.3% (vol/vol) Triton X-100, 1 mM dithiothreitol] containing protease inhibitor cocktail set III (PICSIII; EMD Biosciences, San Diego, CA) for 10 min at 4 C, and extracts were centrifuged to pellet cellular debris. Protein concentrations were determined using the BCA protein assay kit (Pierce, Rockford, IL). Samples (600 µg) were precleared with 1 µl normal mouse or rabbit serum (Jackson Immunoresearch) and 20 µl protein A/G Sepharose beads (Santa Cruz Biotechnology) for 30 min at 4 C. Immunoprecipitation was performed on a 275-µg sample with 2 µg Flag monoclonal antibody (Sigma) or normal mouse serum for 1 h at 4 C. Protein A/G Sepharose beads were added and the samples incubated overnight on a nutator at 4 C. Beads were washed, and 30 µl sample extraction buffer (containing PICSIII) and 30 µl Laemmli sample buffer were added to the beads or to 10 µg total lysate.

SDS-PAGE and Western Blot Analysis
siRNA samples were evaluated for MUC1 protein expression using the 214D4 antibody as described previously (12). A GAPDH monoclonal antibody (Chemicon, Temecula, CA) was added at a dilution of 1:5500 and incubated overnight at 4 C. Membrane was washed with PBS-Tween (1% [vol/vol]), then incubated for 2 h at 4 C with horseradish peroxidase-conjugated sheep antimouse IgG (Jackson ImmunoResearch) at a final dilution of 1:200,000 in blocking solution. SuperSignal West Dura Extended Duration Substrate (Pierce) was used for detection, per the manufacturer’s instructions.

Coimmunoprecipitation samples (25 µl) or 10 µg total lysate were boiled before electrophoresis on a 4–12% (wt/vol) acrylamide NuPage Bis-tris gel (Invitrogen) in 1x MOPS buffer at 200 V for 45 min. Proteins were transferred to nitrocellulose membrane and blocked in PBS-Tween containing 5% (wt/vol) nonfat milk. A PR monoclonal antibody (PgR Ab-8x, Santa Cruz Biotechnology) was added at a dilution of 1:12,500 and incubated overnight at 4 C. Membranes were washed with PBS-Tween, then incubated with horseradish peroxidase-conjugated sheep antimouse IgG and detected as described above. Band intensities were quantitated using the Alpha Imager 1D-Multi Function (Alpha Innotech).

ChIP Assays
ChIP assays were performed as described (12), with the exception that Flag-PIASy or Flag-pCMV5 was transfected into the cells before treatment, as described above. Antibodies to PR (C-20), SRC-1 (M-341), SRC-3 (M-397), SMRT (N-20), and NCoR (H-303) were purchased from Santa Cruz Biotechnology.

Data Analysis
Unless otherwise noted, data are shown as the means ± SEM of triplicate samples and are representative of at least two independent experiments. All data were analyzed by Student’s t test or one-way ANOVA followed by the Tukey-Kramer multiple comparisons test, as appropriate, using the GraphPad InStat version 3.05 software (GraphPad Software, Inc., San Diego, CA).


   ACKNOWLEDGMENTS
 
We thank Dr. Ke Shuai for providing the PIAS expression plasmids, Dr. Sandra Gendler for the MUC1 promoter plasmid, Dr. Pierre Chambon for the hPRA and hPRB plasmids, Dr. Doug Kniss for the HES cell line, Dr. Edward Yeh for the SENP-1 expression plasmid, and Dr. John Hilkens for the 214D4 antibody. We are greatly indebted to Barbara Malone, Ben Rohe, and JoAnne Julian for many helpful discussions. We are grateful to Dr. Pamela Mellon and the University of California, San Diego, where M.J.B. was located for part of this work. We are especially grateful to Doreen Anderson and Sharron Kingston for their expert secretarial assistance and to Dr. Kirk Czymmek for microscopy assistance.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grant UO1 HD 29963 to D.D.C. as part of the National Cooperative Program on Trophoblast-Maternal Tissue Interactions, and National Science Foundation Integrative Graduate Education and Research Traineeship Fellowship (Grant 0221651) to M.J.B.

Disclosure Summary: M.J.B., N.D., and E.L. have nothing to declare. D.D.C. consulted for and received lecture fees from Alcon Laboratories, Inc.

First Published Online August 23, 2007

Abbreviations: ChIP, Chromatin immunoprecipitation; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDAC, histone deacetylase; IFN-{gamma}, interferon-{gamma}; MUC1, mucin 1; NCoR, nuclear receptor corepressor; NF{kappa}B, nuclear factor-{kappa}B; P, progesterone; PR, progesterone receptor; PIAS, protein inhibitor of activated signal transducer and activator of transcription; PRE, progesterone response element; SENP-1, sentrin/SUMO-specific protease-1; siRNA, small interfering RNA; SMRT, silencing mediator of retinoid and thyroid receptors; SRC, steroid receptor coactivator; STAT, signal transducer and activator of transcription; SUMO, small ubiquitin-like modifier; UE, uterine epithelium.

Received for publication December 15, 2006. Accepted for publication August 7, 2007.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   PR
Coregulators:   ARIP3  |  PIAS1  |  PIAS3  |  PIAS4  |  SRC-1  |  AIB1  |  NCOR  |  SMRT
Ligands:   Progesterone  |  R5020



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