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Molecular Endocrinology, doi:10.1210/me.2005-0343
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Molecular Endocrinology 20 (10): 2278-2291
Copyright © 2006 by The Endocrine Society

Progesterone Receptor Isoforms A and B Differentially Regulate MUC1 Expression in Uterine Epithelial Cells

Melissa J. Brayman, JoAnne Julian, Biserka Mulac-Jericevic, Orla M. Conneely, Dean P. Edwards and Daniel D. Carson

Department of Biological Sciences (M.J.B., J.J., D.D.C.), University of Delaware, Newark, Delaware 19716; and Department of Molecular and Cellular Biology (B.M.-J., O.M.C., D.P.E.), Baylor College of Medicine, Houston, Texas 77030

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
 
MUC1 expression responds differently to changes in progesterone (P) levels in mouse vs. human uterine epithelium. Two isoforms of progesterone receptor, PRA and PRB, mediate the physiological effects of P. Using transient transfection of a human uterine epithelial cell line, HEC-1A, we showed that liganded PRB stimulated MUC1 gene activity. PRA alone had little effect on MUC1 promoter activity, but antagonized the PRB-mediated stimulation. The region from 523 to 570 bp upstream of the transcriptional start site was shown to be required for the P response. Mutation of two potential P-responsive element (PRE) half-sites in this region partially inhibited the PRB-mediated response, and one PRE half-site disrupted binding of both PRB and PRA to a consensus PRE in an EMSA. These along with other studies indicated that multiple cis elements in the –523- to –570-bp region cooperate to mediate P responsiveness, and that PR interaction with other transcription factors in this region is likely. Using ovariectomized wild-type, PR knockout (PRKO), PRAKO, and PRBKO mice, P antagonism of estrogen-stimulated Muc1 protein and mRNA expression was shown to be dependent on PRA. In summary, these data show that liganded PRB stimulates MUC1 expression in human uterine epithelial cells, whereas liganded PRA antagonizes MUC1 expression in both human and mouse uterine epithelial cells. The differential MUC1 response to P in these two species may be due to dissimilar expression of the two PR isoforms in the uterine epithelium.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE MUC1 GENE encodes a highly glycosylated, transmembrane glycoprotein that is expressed on the apical surface of most normal simple epithelia, including those of the mammary gland, female reproductive tract, lung, kidney, stomach, gall bladder, and pancreas (1), and in some nonepithelial cell types. The apical glycocalyx, of which MUC1 is a part, serves to lubricate and hydrate mucosal tissues, as well as to protect epithelial surfaces from microbial attack (2). In the uterus, evidence suggests that MUC1 has the potential to prevent interaction between an implanting embryo and the epithelial cell surface, thereby preventing implantation (3, 4). Based on studies in animal and in vitro model systems, it is likely that MUC1 must be removed in order for implantation to occur. In fact, Muc1 (mouse nomenclature) expression in mice is lost throughout the uterine epithelia during the window of receptivity on d 4 of pregnancy, when the uterus is receptive to blastocyst attachment (5, 6). In contrast, uterine luminal expression of Muc1 remains high during the receptive phase in rabbits, with a local loss of Muc1 protein at implantation sites (7). Likewise, MUC1 expression in humans is high during the receptive phase (8, 9).

In both mice and humans, progesterone (P) levels are maximal, relative to estrogen (E2), at the implantation phase, but MUC1 regulation by steroid hormones is different in the two species. In mice, Muc1 expression in the uterus is stimulated by E2 and down-regulated by P (5, 6), although no direct regulation of the Muc1 promoter by the estrogen receptors, ER-{alpha} and ER-ß, or progesterone receptors, PRA and PRB, has been shown (Ref. 10 ; Zhou, X., and D. Carson, unpublished results). Conversely, endometrial MUC1 expression in humans is higher during the secretory phase, a progesterone-dominated portion of the cycle (9, 11). Previous studies have examined the regulation of human MUC1 expression by steroid hormones in a number of cell types (12, 13, 14), but no direct regulation by the ERs or PRs has been shown.

Steroid hormonal regulation of gene expression in the uterus is a complex process mediated through the ERs and PRs. E2 and P have diverse effects on responsive tissues due, in part, to the presence of the different isoforms of each receptor. Moreover, various cofactors are present in different cell types that can associate with transcription factors to modulate their activity (15). The PRA and PRB isoforms are products of a single gene with two transcriptional start sites. The PRB protein contains 164 amino acids at its amino terminus that are not found in PRA (16, 17, 18). This corresponds to a transactivation region that can interact with coactivators that do not effectively bind PRA, whereas PRA has a higher affinity for corepressors than PRB, giving the two receptors dissimilar transactivation functions (19). PRA is able to transrepress PRB-mediated transcriptional activation when both isoforms are coexpressed (20, 21). PRA knockout (PRAKO) mice show increased proliferation of the uterine epithelium, demonstrating that PRA represses P- and E2-mediated cell proliferation (22). PRBKO mice, on the other hand, have almost normal uterine morphology (21). This suggests that PRA is the dominant PR isoform in the mouse uterus. On the other hand, PRB is highly expressed in human uterine epithelia in the early to midsecretory phase, after which time total PR levels decrease and the PRA:PRB ratio increases (23, 24, 25).

In this study, we demonstrate that liganded PRA and PRB differentially regulate MUC1 gene expression in a human uterine epithelial cell line, HEC-1A, in vitro, as well as in mouse uterine epithelia in vivo. It is likely that the differences in P regulation of MUC1 expression among species are due, in part, to differences in the dominance of PR isoforms in uterine epithelia.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Progesterone, But Not Estrogen, Stimulates MUC1 Promoter Activity
To examine the effect of P on MUC1 promoter activity in human uterine epithelial cells, HEC-1A cells were transiently cotransfected with a human PRB expression plasmid (hPRB) and either a P-responsive element (PRE)- or MUC1 promoter-driven luciferase reporter construct (PRE and 1.4MUC, respectively). Cells were treated with or without 400 nM P for 24 h. Cotransfection of the hPRB and PRE plasmids resulted in approximately 4-fold stimulation of luciferase activity in response to P, and cotransfection of the hPRB and 1.4MUC plasmids resulted in a 3.2-fold stimulation of luciferase activity in response to P (Fig. 1AGo). The P-induced induction of both PRE and 1.4MUC promoter activity was completely reversed by treatment with 400 nM RU486. No response to P was seen in HEC-1A cells transfected with 1.4MUC and an empty vector (data not shown). Similar results were obtained with another human uterine epithelial cell line, HES (Dharmaraj, N., and D. D. Carson, unpublished results). These data indicate that MUC1 promoter activity is stimulated by P in a PRB- and ligand-dependent manner in HEC-1A cells.


Figure 1
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  		Fig. 1. Liganded PRB, But Not ER-{alpha}, Stimulates MUC1 Promoter Activity in HEC-1A Cells

A, HEC-1A cells were transiently transfected with hPRB and a reporter construct containing either a PRE upstream of the thymidine kinase (TK) promoter (PRE), or the 1.4MUC construct, and then treated with 0.002% (vol/vol) ethanol (open box), 400 nM P (black box), or 400 nM P plus 400 nM RU486 (hatched box) for 24 h. B, HEC-1A cells were transiently transfected with an ER{alpha} expression plasmid and a reporter construct containing either three EREs upstream of the TK promoter (ERE), or the 1.4MUC promoter, and then treated with 0.001% (vol/vol) ethanol vehicle, or 1 nM E2 for 24 h. Data are expressed as the mean ± SEM, relative to vehicle-treated, and are representative of at least two independent experiments performed in triplicate. *, P < 0.01; **, P < 0.001 vs. vehicle treated.

 
To examine the effect of another steroid hormone receptor on MUC1 promoter activity in human uterine epithelial cells, HEC-1A cells were transiently transfected with an ER-{alpha} expression plasmid and either the ERE or 1.4MUC reporter construct, and treated with or without 1 nM E2 for 24 h. E2 treatment of HEC-1A cells containing the ERE reporter resulted in increased luciferase activity (~2.5-fold), but E2 treatment had no effect on activity from the 1.4MUC reporter construct (Fig. 1BGo). In contrast to PRB, these data suggest that liganded ER-{alpha} does not directly regulate MUC1 promoter activity in HEC-1A cells.

HEC-1A cells were transiently cotransfected with hPRB and the 1.4MUC reporter construct, and then treated with increasing concentrations of P for 24 h. A threshold effect was seen (Fig. 2Go); 400 nM P was the lowest dose at which significant stimulation of MUC1 promoter activity is seen. This is a physiologically relevant concentration because circulatory P levels in women at the receptive phase can be as high as 60 nM (26), and endometrial concentrations may be as much as 10-fold higher than plasma concentrations (27).


Figure 2
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  		Fig. 2. Dose-Dependent Stimulation of MUC1 Promoter Activity by P

HEC-1A cells were transiently transfected with hPRB and 1.4MUC, and then treated with 0.001% (vol/vol) ethanol vehicle or the indicated concentrations of P for 24 h. Data are expressed as the mean ± SEM, relative to vehicle-treated, and are representative of at least two experiments performed in triplicate. ***, P < 0.001 vs. 0 nM P.

 
MUC1 Protein Expression Is Stimulated by P in HEC-1A Cells Stably Expressing PRB
To determine whether P is able to stimulate MUC1 protein expression, HEC-1A cells were stably transfected with hPRB. Stably transfected clones were screened for PRB expression by Western blotting and transient transfection assays with a consensus PRE reporter (data not shown). PRB3 and PRB11, two clones that stably expressed PRB, and the parental (HEC-1A) cells were treated with P or vehicle for 48 h. Cell-associated MUC1 was analyzed by Western blotting (Fig. 3AGo). Whereas the parental cells showed no change in MUC1 protein expression in response to P, both PRB-expressing cell lines displayed an approximately 2-fold increase in cell-associated MUC1 in response to P treatment (Fig. 3BGo). There is a similar increase in MUC1 released into the medium upon treatment with P (data not shown).


Figure 3
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  		Fig. 3. P Stimulates MUC1 Expression in HEC-1A Cells Stably Expressing PRB

A, The parental cell line or the stably transfected clones, PRB3 or PRB11, were treated with 0.001% (vol/vol) ethanol vehicle or 400 nM P for 48 h and all extracts were examined by Western blotting for MUC1. B, The bar graphs represent the intensities of the MUC1 bands, determined by densitometry. Data are expressed as the mean ± SEM, relative to vehicle-treated, and are representative of at least two independent experiments performed in triplicate. **, P < 0.01; ***, P < 0.001 vs. vehicle-treated.

 
PRB-Mediated P Stimulation of MUC1 Promoter Activity Is Antagonized by PRA
To determine whether PRA also modulated MUC1 promoter activity, HEC-1A cells were transiently transfected with the PRA expression plasmid (hPRA) and the 1.4MUC construct, and treated with increasing concentrations of P for 24 h (Fig. 4AGo). A slight stimulation (up to 1.4-fold) of MUC1 promoter activity was observed at 40 and 400 nM P. When the cells were transiently transfected with 1.4MUC, hPRB, and increasing amounts of hPRA, PRA inhibited PRB-stimulated promoter activity in a PRA dose-dependent fashion (Fig. 4BGo). These data suggest that PRA acts as a transrepressor of PRB action in this context.


Figure 4
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  		Fig. 4. PRA Inhibits PRB-Mediated P Stimulation of MUC1 Promoter Activity

A, HEC-1A cells were transiently transfected with 1.4MUC and hPRA, and then treated with 0.001% (vol/vol) ethanol vehicle or the indicated P concentrations for 24 h. *, P < 0.05; **, P < 0.01 compared with vehicle alone. B, HEC-1A cells were transiently transfected with 1.4MUC, 2 µg hPRB, and indicated amounts of hPRA, and then treated with 0.001% (vol/vol) ethanol vehicle or 400 nM P for 24 h. Data are expressed as the mean ± SEM, relative to vehicle-treated, and are representative of at least two independent experiments performed in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. the immediately adjacent group on the left.

 
PRB-Mediated P Induction of MUC1 Promoter Activity Requires the PRB DNA Binding Domain (DBD)
The classical mechanism of PR action on a promoter requires PR binding to a PRE via its DBD, and subsequent recruitment of cofactors to activate or repress transcription (20). Recently, it has been shown that PR can interact with other transcription factors to regulate transcription in a PRE-independent manner (28, 29). To determine whether DNA binding is required for the PRB-mediated P response of the MUC1 promoter, HEC-1A cells were transiently transfected with 1.4MUC and hPRB and/or an expression plasmid containing PRB with a mutation in the DBD (mutPRB), then treated with or without 400 nM P for 24 h. Little or no induction of MUC1 promoter activity was seen in the cells transfected with mutPRB alone, and mutPRB completely blocked PRB-mediated stimulation (Fig. 5AGo). These data suggested that the PRB DBD is required for P induction of MUC1 promoter activity, and that the DBD mutation either disrupts direct PRB binding to DNA and/or interferes with PRB interaction with another factor(s) that can tether PRB to the promoter region. The p21 promoter is stimulated by P in the T47D breast carcinoma line, and this stimulation requires PRB interaction with Sp1 in a PRE-independent manner (30). Indeed, liganded mutPRB was able to activate transcription from the p21 promoter in the T47D cell line (data not shown), suggesting that the DBD mutation does not disrupt the PRB-Sp1 interaction in this context.


Figure 5
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  		Fig. 5. The DBD Is Required for PRB Activation, But Not PRA Transrepression, of MUC1 Promoter Activity

A, HEC-1A cells were transiently transfected with 1.4MUC, and hPRB alone, both hPRB and mutPRB, or mutPRB alone and treated with 0.001% (vol/vol) ethanol vehicle or 400 nM P for 24 h. B, HEC-1A cells were transiently transfected with 1.4MUC, and hPRB alone, hPRB and hPRA, or hPRB and mutPRA and treated with 0.001% (vol/vol) ethanol vehicle or 400 nM P for 24 h. Data are expressed as the mean ± SEM, relative to vehicle-treated, and are representative of at least two independent experiments performed in triplicate. ***, P < 0.001 vs. hPRB alone.

 
PRA Transrepression of PRB-Mediated Activity Does Not Require the PRA DBD
PRA transrepression of nuclear receptors, including ER-{alpha} and PRB, has been shown to be independent of DNA binding (31). To determine whether DNA binding is required for PRA-mediated transrepression of PRB activity in our system, HEC-1A cells were transiently transfected with 1.4MUC, hPRB, hPRA, and/or and expression plasmid containing hPRA with a mutation in the DBD (mutPRA), and then treated with or without 400 nM P for 24 h. PRB stimulation of MUC1 promoter activity was repressed both by wild-type and mutant PRA (Fig. 5BGo), suggesting that the PRA DBD is not required for transrepression of PRB. Alternatively, repression may be due to the formation of nonfunctional mutPRA:PRB heterodimers.

P Responsiveness of the MUC1 Promoter Lies within the Region from 570 to 523 bp Upstream of the Start Site of Transcription
To locate the P-responsive region of the 1.4 kb MUC1 proximal promoter, a series of 5' deletion mutants were tested in transient transfection assays of HEC-1A cells cotransfected with hPRB. Maximal P response (4.5-fold) was found to reside within the proximal 570 bp upstream of the MUC1 transcriptional start site (570MUC), although basal transcription from this construct is reduced, probably due to the loss of the Sp1 site at –576/–568 (32). Further deletion from –570 to –523 (523MUC) resulted in a loss of most of the P response (Fig. 6AGo). To determine whether this region was sufficient to confer P responsiveness to an enhancerless promoter, a heterologous reporter construct containing the –604 to –468 promoter region upstream of the thymidine kinase promoter (604/468-TK) was used. This heterologous promoter includes the –589/–580 {kappa}B site, the –576/–568 Sp1 site, the P-responsive region, and the –503/–495 signal transducer and activator of transcription (STAT) binding site, and also was responsive to P (3.1- vs. 1.6-fold for pGL3-TK). This response was antagonized by coexpression of PRA (Fig. 6BGo). A heterologous promoter construct containing the –564 to –500 promoter region (lacking the {kappa}B, Sp1, and STAT binding sites) also retained P responsiveness similar to that observed from 1.4MUC (data not shown). Although some P responsiveness (~2-fold) was retained in the –523 to +33 region of the MUC1 proximal promoter, it is not clear whether this response is specific to the MUC1 promoter or to the pGL3 vector (pGL3-basic). We concluded that the MUC1 proximal promoter region from 570 to 523 bp upstream of the transcription start site is required for PRB stimulation, as well as PRA antagonism of PRB action.


Figure 6
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  		Fig. 6. P-Responsiveness Is Located between 523 and 570 bp Upstream of the MUC1 Transcriptional Start Site

A, HEC-1A cells were transiently transfected with the indicated deletion reporter constructs of the 1.4MUC promoter construct and hPRB. Cells were treated with 0.001% (vol/vol) ethanol vehicle (open boxes) or 400 nM P (black boxes) for 24 h before assay. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 vs. 1.4MUC. Data are expressed as the mean ± SEM, and are representative of at least two independent experiments performed in triplicate. Fold P response is the fold increase in promoter activity upon treatment with P relative to vehicle-treated. B, HEC-1A cells were transiently transfected with a heterologous promoter of the –468- to –604-bp MUC1 proximal promoter region, fused upstream of a basal thymidine kinase promoter, and cotransfected with hPRB, hPRA, or both hPRA and hPRB as indicated. Cells then were incubated with 0.001% (vol/vol) ethanol vehicle or 400 nM P for 24 h before assay. ***, P < 0.001 vs. hPRB alone. Data are expressed as the mean ± SEM, relative to vehicle-treated, and are representative of at least two independent experiments performed in triplicate.

 
To further characterize the cis-elements in the MUC1 promoter responsible for PR responsiveness, the proximal 570-bp region of the MUC1 promoter was analyzed using the Transcription Element Search Software (33). The sequence analyzed has been published previously (34) and was verified in the 1.4MUC plasmid by DNA sequencing. Five potential PRE half-sites were identified in this region: PRE1 (5'-AGAACT-3' located at –552 to –547), PRE2 (5'-GGGACA-3' located at –535 to –530), PRE3 (5'-TGTACA-3' located at –352 to –347), PRE4 (5'-AGAACA-3' located at –196 to –191), and PRE5 (5'-TGTGCC-3' located at –67 to –62).

To determine the potential role of these elements in regulating expression from the MUC1 gene, the putative PREs were mutated singly and in combination. Mutation of PRE1 had no effect on the PRB-mediated response, but significantly reduced basal expression from the MUC1 promoter (mutPRE1; Fig. 7AGo). Mutation of both PRE1 and PRE2 resulted in a 33% reduction in PRB-mediated response to P, and further repressed basal expression (mutPRE1–2; Fig. 7AGo). We hypothesized that one or more of the three most proximal PREs, i.e. PRE3, PRE4, and PRE5, might be responsible for the residual P stimulation seen in the –523MUC, –487MUC, and –169MUC deletion constructs; however, no change in P responsiveness was seen when these PREs were mutated alone (data not shown) or in combination (mutPRE3–5; Fig. 7BGo), suggesting that the PRB is acting through a cis-element other than the consensus PREs found in this region, or that the vector itself confers some P responsiveness.


Figure 7
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  		Fig. 7. PRE1 and PRE2 Are Partially Responsible for the P-Responsiveness of the MUC1 Promoter

HEC-1A cells were transiently transfected with hPRB and 1.4MUC containing mutations in (A) PRE1 (mutPRE1) or PRE1 and PRE2 (mutPRE1–2), (B) PRE3, PRE4 and PRE5 (mutPRE3–5), the Sp1 site at –99/–90 (mut90Sp1), or at –576/–567 (mut576Sp1). Cells were treated with 0.001% (vol/vol) ethanol vehicle (open boxes) or 400 nM P (black boxes) for 24 h before assay. Data are expressed as the mean ± SEM and are representative of at least two independent experiments performed in triplicate. Fold P response is the fold increase in promoter activity upon treatment with P relative to vehicle treated. **, P < 0.01; and ***, P < 0.001 vs. 1.4MUC.

 
The Sp1 family of transcription factors has been shown to interact with nuclear receptors, including PR, to mediate steroid hormone action in the uterine endometrium (30, 35, 36). These interactions may require a GC-rich domain, a PRE, or both. We examined the possible role of the Sp1 binding sites in the MUC1 promoter on P responsiveness. Two functional Sp1 sites reside in the MUC1 proximal promoter region at –576/–568 and –99/–90 (32, 37, 38). As has been shown previously, mutation either site severely reduced basal activity (32); however, mutagenesis of either Sp1 site had no effect on liganded PRB stimulation of MUC1 promoter activity (Fig. 7BGo), suggesting that PRB does not act through these Sp1 sites to regulate MUC1 promoter activity. This is consistent with the data from the deletion constructs, because constructs lacking either the –576/–568 or –99/–90 site (570MUC and 604/468TK, respectively) or both sites (564/500-TK; data not shown) retained P responsiveness.

Collectively, these data were consistent with the results of deletion and heterologous promoter analyses that mapped the PR-responsive element(s) between –570 and –523; however, mutations of any likely candidate elements, singly or in combination, in this region resulted in only partial reduction in P-mediated activity. Thus, we concluded that this region is comprised of several additive enhancer modules that also contribute to basal promoter activity. We have not ruled out the possibility that PRB interaction with another transcription factor(s) in this region is required for the P response.

Binding of PR to the Putative PRE
EMSAs were performed with a synthetic oligonucleotide containing a consensus PRE in competition with wild-type and mutated consensus PRE, PRE1, the –570 to –523 region of the MUC1 promoter (570/523), or an antibody against PR (Fig. 8Go). Strong specific DNA-protein complexes were observed upon addition of insect cell extracts containing either PRA (Fig. 8AGo, lane 2) or PRB (Fig. 8BGo, lane 2). This complex was supershifted upon addition of anti-PR antibody (lane 3) but not preimmune rabbit IgG (lane 4). The DNA-protein complex was eliminated almost completely upon addition of excess competitor DNA containing an unlabeled consensus PRE (lane 5), PRE1 (lane 7), or the 570/523 fragment (lane 9), but not by a mutated consensus PRE (lane 6), mutated PRE1 (lane 8), or either Sp1 site (–576/–568 or –99/–90; data not shown). These results suggest that PRE1 can bind both isoforms of PR and is likely to be at least partially responsible for PR-mediated regulation of the MUC1 promoter; however, we cannot rule out the possibility that PR interaction with another transcription factor is required for PR interaction with the MUC1 promoter in vivo.


Figure 8
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  		Fig. 8. The PRE1 Probe, But Not a Mutant PRE1, Inhibits PR Binding to a Consensus PRE

A synthetic oligonucleotide containing the consensus PRE was biotin-labeled and incubated with WCE from insect cells expressing PRA (A) or PRB (B) that had been treated with 200 nM R5020 for 24 h. Specific DNA-protein complexes (indicated by arrow) were seen upon addition of WCE (lane 2) and were supershifted by the antibody against PR (lane 3), but not rabbit IgG (lane 4). DNA-protein complexes were disrupted in the presence of unlabeled competitor PRE (lane 5), PRE1 (lane 7), or the –570 to –523 region of the MUC1 promoter (lane 9), but not mutated consensus PRE (lane 6) or mutated PRE1 (lane 8).

 
Recruitment of PRA and PRB to the MUC1 Proximal Promoter
Chromatin immunoprecipitation (ChIP) assays were used to determine whether the PR isoforms interact with the MUC1 promoter region in vivo. HEC-1A cells were transiently transfected with hPRA, hPRB, or both and treated with 400 nM P. Both PRA and PRB were recruited to the MUC1 P-responsive promoter region (–618/–500), but not a control region (–163/–23), when expressed alone (Fig. 9AGo), but less recruitment is seen when the two isoforms were coexpressed (Fig. 9Go, A and B). Untransfected HEC-1A cells show no PR interaction in these promoter regions (data not shown). These data suggest that both PRA and PRB interact with the MUC1 promoter in this region, but PRA interferes with PRB binding to the promoter. The difference in regulation of the MUC1 promoter by these isoforms likely is due both to differential cofactor binding as well as differential recruitment to the promoter region.


Figure 9
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  		Fig. 9. PRs Are Recruited to the P-Responsive Region of the MUC1 Promoter in Vivo

HEC-1A cells were transiently transfected with hPRA, hPRB, or both hPRA and hPRB. Twenty-four hours after transfection, cells were treated with 400 nM P for 1 h, cells were cross-linked with 1% formaldehyde, soluble chromatin was extracted, and ChIP assays were performed using no antibody or anti-PR antibody. A, PCR was performed with primers specific to the P-responsive region (–618/–500) or control region (–163/–23) of the MUC1 promoter. Input lane represents one fourth total chromatin. B, Real-time PCR was performed with primers specific to the P-responsive region. Data are presented relative to input chromatin, as a percentage of PRB. Data are representative of at least three independent experiments. **, P < 0.01 vs. PRB.

 
PRA, But Not PRB, Is Responsible for Repression of the E2-Mediated Increase in Muc1 by P in Mouse Uterine Epithelial Cells in Vivo
To examine the correlation of PR isoform expression on Muc1 regulation by steroid hormones in an in vivo context, wild-type, PRKO, PRAKO, and PRBKO mice were ovariectomized and treated with oil, E2 alone, or E2 and P, for 3 d. In wild-type mice, ovariectomy caused a complete reduction in Muc1 protein expression throughout the luminal epithelia (LE) and glandular epithelia (GE) by 18 d after surgery (Fig. 10AGo). As expected (6), E2 treatment caused a dramatic increase in MUC1 protein levels in both the LE and GE (Fig. 10BGo), and treatment with P in addition to E2 attenuated the E2 response in this regard (Fig. 10CGo). In contrast, Muc1 expression persisted in the GE of ovariectomized PRKO mice (Fig. 10DGo), and expression was stimulated both by treatment with E2 alone (Fig. 10EGo) as well as E2 and P (Fig. 10FGo), indicating that the attenuation of Muc1 response to E2 by P requires PR. The Muc1 expression pattern in PRBKO uteri (Fig. 10Go, G–I) mimicked that of wild-type mice, indicating that PRB is not required for P-dependent attenuation of Muc1 expression. In contrast, the Muc1 expression pattern in PRAKO uteri (Fig. 10Go, J–L) was similar to that observed in PRKO mice, indicating that PRA is essential for this P response. The relative concentrations of Muc1 mRNA extracted from whole uteri of these animals reflected the pattern observed for Muc1 protein expression (Fig. 11Go). Collectively, these data demonstrate that the attenuation of E2-mediated stimulation of Muc1 protein and mRNA expression in mouse uterine epithelia requires liganded PRA, but not PRB.


Figure 10
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  		Fig. 10. PRA Is Required to Repress E2-Mediated Stimulation of Muc1 Protein Expression in Vivo

Anti-Muc1 immunofluorescence was performed on wild-type (WT) (A–C), PRKO (D–F), PRAKO (G–I), or PRBKO (J–L) endometrium. Mice were ovariectomized and treated with oil (A, D, G, and J), 100 ng E2 alone (B, E, H, and K), or 100 ng E2 and 1 mg P (C, F, I, and L) each day for 3 d before harvest and sectioning.

 

Figure 11
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  		Fig. 11. PRA Is Required to Repress E2-Mediated Stimulation of Muc1 mRNA Expression in Vivo

Real-time PCR was performed on mRNA isolated from whole uteri of ovariectomized WT, PRKO, PRAKO, or PRBKO mice, treated with oil, 100 ng E2 alone, or 100 ng E2 and 1 mg P each day for 3 d. Each treatment represents pooled total RNA from five mice. Abundance of MUC1 is expressed relative to K18 by the {Delta}{Delta}Ct method, normalized to GAPDH. Data are expressed as the mean ± SEM of triplicate determinants. ***, P < 0.001 vs. wild type.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
A number of studies have shown that human MUC1 mRNA and protein levels in the uterus fluctuate during the menstrual cycle. MUC1 levels are higher during the receptive phase than in the nonreceptive phase (8, 9, 11). These data suggest that MUC1 expression is regulated by the steroid hormones E2 and P; however, little is known about the role of steroid hormone receptors in regulating MUC1 gene expression in the uterus. In the present study, we have shown that PRA and PRB differentially regulate MUC1 gene transcription in a human uterine epithelial cell line, HEC-1A. Liganded PRB stimulated MUC1 promoter activity, a response that was antagonized by coexpression of PRA. Liganded PRB also stimulated an increase in MUC1 protein levels when stably transfected into the HEC-1A cell line. On the other hand, liganded ER-{alpha}, the predominant ER in the uterus (39), had no effect on MUC1 promoter activity, and E2 treatment was not required for the PRB-mediated response. The latter is not surprising because the hPRB expression plasmid contains a constitutive promoter (40), rather than the PR promoter, which normally requires E2 priming to up-regulate PR expression (41).

The P responsiveness of the MUC1 gene was mapped to the region from 523 to 570 bp upstream of the transcription start site, and both PRA and PRB interact with this region in ChIP assays and EMSA. A 33% reduction in liganded PRB-mediated promoter activity was observed upon mutation of both PRE1 and PRE2, suggesting that these putative PREs are involved in the P responsiveness of the MUC1 promoter. Although mutating PRE1 alone did not significantly reduce the P response, this PRE was able to compete with a consensus PRE for PRA and PRB binding, further supporting its role in the P response. PRs can regulate transcription either by direct binding to a PRE, or by interaction with another transcription factor in a PRE-independent manner (28, 29). Our data suggest that P stimulation of MUC1 promoter activity is complex and that PR interaction with other transcription factors and/or coregulators may stabilize PR interaction with the MUC1 promoter region. It also is possible that the mutated PRE1 sequence was not sufficiently divergent from a PRE sequence to disrupt PR-DNA binding when PR interacts with other factors at the MUC1 promoter.

A PRB containing a mutated DBD antagonized the PRB-mediated MUC1 promoter response, suggesting that DNA binding by PRB, perhaps to a PRE, is required. Although the DBD mutation may have prevented an interaction between PRB and another transcription factor that is required for PRB recruitment to and transactivation of the MUC1 promoter, this is unlikely because the DBD mutation did not affect PRB interaction with Sp1. Interestingly, PRA transrepression of PRB-mediated activation did not require the DBD, an observation that has been reported previously in other contexts (31); however, we do not rule out the possibility of formation of nonfunctional mutPRA:PRB heterodimers. In fact, coexpression of PRA and PRB leads to less binding of the promoter by PR, further suggesting that PRA transrepression is not dependent on DNA binding. Although the PRA DBD is not required for transrepression of PRB, it is likely to be required for the slight activation in promoter activity seen with PRA alone, because liganded PRA binds to the MUC1 promoter region when no PRB is present.

Most mutations that we made in the –570/–523 region resulted in a significant decrease in basal promoter activity, suggesting that this region is very important for MUC1 expression. Indeed, the –570- to –487-bp region of the human MUC1 promoter is 59.5% homologous to the mouse promoter. All of the transcription element binding sites near this region that have been shown to be important for MUC1 expression, namely the Sp1 site at –576/–567 (32), the NF{kappa}B site at –589/–580 (42), and the STAT binding element at –503/–495 (42, 43), are highly conserved between the two species. Systematic point mutations in the analogous region of the murine Muc1 promoter only partially reduced the responsiveness of the promoter to PPAR{gamma} (44), suggesting that this region includes a number of enhancer modules that are important for transcriptional regulation of Muc1 expression. Ongoing experiments are being performed to elucidate potential PR interaction with other transcription factors and cofactors in the –570 to –523 region of the human MUC1 promoter.

The function of nuclear receptors, including PR, is a complex and dynamic system that is highly dependent on the cell type, cellular constituents, and extracellular environment (29, 31, 45). In addition to the promoter sequence, the presence of coactivators, corepressors, other transcription factors, and various extracellular signals can influence the P responsiveness of a gene. The HEC-1A cell line was derived from malignant cells, and as such is certain to differ significantly from normal uterine epithelial cells. However, there is evidence for PR regulation of MUC1 expression in normal human uterine epithelium. Total PR expression in the LE increases through the proliferative phase and peaks at the midsecretory phase, and then decreases to almost undetectable levels by the receptive phase (25, 46, 47). Although PRA is always expressed at a higher level than PRB in the human uterus, the A:B ratio is lowest at the peak of PR expression (48, 49) when MUC1 levels also are high (8, 9), suggesting that PRB may activate MUC1 expression in vivo as well as in vitro. However, the presence of other signaling molecules, e.g. cytokines and growth factors, also regulate MUC1 expression in the LE (50), and stromal PR may play an indirect role in P regulation of MUC1.

Murine Muc1 expression in the receptive uterus differs dramatically from that in human. Muc1 protein is present throughout the LE and GE through d 3 of pregnancy, but is almost completely lost by d 4, a phenomenon that is reversed by treatment with an antiprogestin, RU486 (6). Thus, murine Muc1 expression shows a negative correlation with PR function. As has been shown previously (6), E2 stimulated Muc1 expression in the LE and GE of wild-type mice, and P antagonized this up-regulation. The PRBKO mouse uteri showed a similar pattern of Muc1 expression. In contrast, in PRKO and PRAKO uteri, the E2-mediated Muc1 response was not antagonized by P treatment. Thus, we concluded that PRA, but not PRB, is required for the P-mediated repression of Muc1 expression in mouse uterine epithelial cells in vivo.

Taken together, these data suggest that the differences observed in human vs. murine MUC1 expression in the uterus may be due to differences in PR isoform expression between these two species. Indeed, PRA is the predominant isoform in the mouse uterus (21, 22), and PRB is most abundant in the human uterus during the midsecretory phase (23, 24). A difference in cellular context, including cofactors and other transcription factors, also may explain the species-dependent differences in MUC1 expression. These observations do not rule out the possibility of indirect hormonal regulation of Muc1 expression, possibly though stromal receptors, a scenario that is likely for E2 regulation of Muc1 because the murine promoter is not directly responsive to E2 (10). Because the E2 stimulation of Muc1 protein levels in the uterine epithelium appears to require stromal ER, P ablation of this response cannot be examined in a single cell line. To this end, a uterine stromal cell-specific PRA knockout mouse model would be valuable in sorting out the regulation of murine Muc1 expression by PRA.

Several genes have been shown to be differentially regulated by the two PR isoforms in a promoter- and tissue-specific manner (31, 51, 52, 53); these differences are due, at least in part, to distinct cofactor binding, with PRA having a higher affinity for corepressors than PRB (19). In addition, PR-mediated transrepression of nuclear receptors, including PRB and ER-{alpha}, requires sumoylation of the PR N-terminal region (54). Further studies are required to examine differences in cofactor recruitment to the MUC1 promoter upon binding of PRB, PRA, or both, and whether sumoylation plays a role in PRA transrepression of PRB. The MUC1 gene offers an endogenous promoter model that may help to elucidate the interactions between the two PR isoforms, including differential cofactor binding, in a physiologically relevant context.

In summary, we have shown that MUC1 gene expression is differentially regulated by PRA and PRB in both human and mouse uterine epithelium. PRB stimulates human MUC1 promoter activity, and PRA antagonizes this response, whereas in the mouse PRA is the functional antagonist of E2-stimulated Muc1 expression. This differential regulation may explain the disparity between murine and human MUC1 expression during the receptive period. Potential explanations for infertility may include an overabundance of MUC1 in the LE that inhibits embryo implantation, and/or the inability to down-regulate or clear MUC1 from the epithelial cell surface. Therefore, knowledge of how MUC1 expression can be down-regulated in human uterine LE may aid in vitro fertilization protocols by decreasing MUC1 expression and increasing the availability of the uterine luminal cell surface to the embryo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture
HEC-1A cells were purchased from American Type Culture Collection (Manassas, VA). Cells were maintained in phenol red-free DMEM-F12 medium (Invitrogen, Carlsbad, CA), supplemented with 10% (vol/vol) charcoal-stripped fetal bovine serum (FBS) (HyClone, Logan, UT).

Generation of Plasmid Constructs
The 1.4MUC promoter construct was the generous gift of Dr. Sandra Gendler (Mayo Clinic, Scottsdale, AZ). The human ER-{alpha} expression plasmid and ERE reporter were generous gifts from Dr. John T. Koh (University of Delaware, Newark, DE). The hPRB and hPRA expression plasmids were generous gifts from Dr. Pierre Chambon (College de France, Cedex, France) (40). The mutPRB expression plasmid was the generous gift of Dr. Randal C. Jaffe (University of Illinois, Chicago, IL), and contains a mutation of the GSCKV of the DBD to AACKV. The mutPRA plasmid was created using the QuikChange II Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA), with the same mutation as mutPRB. The 604MUC, 570MUC, 487MUC, pGL3-TK, 604/468MUC, and 518/480MUC plasmids were made as described (42). The 523MUC plasmid was generated by PCR; insert consisting of the MUC1 promoter fragment –523/+33 was cloned into pGL3-basic (Promega, Madison, WI). The PRE reporter construct was made previously by cloning a PRE fragment (55) into the pGL3-TK vector. The 169MUC construct was made using the Erase-a-Base System (Promega), following the manufacturer’s instructions. The extent of digestion was determined by sequence analysis (Cornell DNA Sequencing, Ithaca, NY). The mutant PRE and Sp1 constructs were made using either the GeneTailor Site-Directed Mutagenesis System (Invitrogen) or the QuikChange II Site-Directed Mutagenesis kit following the manufacturers’ instructions, using the 1.4MUC construct as a template. Mutagenic oligos were synthesized by Sigma-Genosys (The Woodlands, TX). The native putative PRE sequence at –61 (PRE5) is 5'-TGTGCC-3', at –196 (PRE4) is 5'-AGAACA-3', at –347 (PRE3) is 5'-TGTACA-3', at –530 (PRE2) is 5'-GGGACA-3', and at –547 (PRE1) is 5'-AGAACT-3'; the mutated sequence for all five is the EclXI recognition sequence, 5'-cGgcCg-3' (mutated nucleotides in lowercase). The native Sp1 sequence at both –90 and –576 is 5'-GGGGCGGGG-3' (32) and the mutant sequence is 5'-GGttCGGGG-3'. Mutation was confirmed by restriction digest and sequence analysis.

Transient Transfections and Reporter Assays
HEC-1A cells were plated in six-well plates that had been coated with growth factor-reduced Matrigel matrix (BD Biosciences, San Jose, CA), and maintained as described until cells reached 60–75% confluence. Cells then were serum-starved for 24 h before transient transfection. Transient transfections were performed using LipofectAMINE reagent (Invitrogen) per the manufacturer’s instructions. Two micrograms of each expression and reporter plasmid (unless otherwise noted), 0.25 µg of pRL-TK plasmid, and 12.5 µl of LipofectAMINE reagent were used per well. For transient transfections where more than one expression vector is added per well, the pSG5 empty vector was added to comparative wells with only one expression vector to keep total plasmid DNA added per well constant. Five hours after transfection, transfection medium was removed and cells were allowed to recover in fresh medium containing 1% (vol/vol) charcoal-stripped FBS for 12 h. Progesterone (Sigma, St. Louis, MO), estrogen (Sigma), RU486 (Roussel UCLAF, Paris, France), and/or ethanol were added in serum-free DMEM-F12 for 24 h. The Dual-Luciferase Assay kit (Promega) was used to lyse the cells and luciferase assays were performed according to the manufacturer’s instructions; activity was measured using a Dynex MLX Microplate Luminometer (Dynex Technologies, Chantilly, VA). Reporter activity was expressed as the ratio of firefly luciferase activity to Renilla luciferase activity.

Stable Transfections
HEC-1A cells were cotransfected with the hPRB expression and pcDNA3.1 (Invitrogen) plasmids using LipofectAMINE reagent per the manufacturer’s instructions. Cells were selected and maintained in DMEM-F12 containing 10% (vol/vol) charcoal-stripped FBS and 500 ng/ml Geneticin (Invitrogen). Stably transfected clones were identified by Western blotting for PRB using the PgR Ab-8 antibody (NeoMarkers, Fremont, CA), and by transient transfection with the PRE reporter construct, with and without 400 nM P treatment.

Western Blot Analysis
HEC-1A cells were plated in six-well plates that had been coated with growth factor-reduced Matrigel matrix, and maintained as described until cells reached 60–75% confluence. Cells were serum-starved for 24 h before treatment. Cells then were treated as described, lysed with sample extraction buffer [SEB; 0.05 M Tris (pH 7.0), 8 M urea, 1.0% (wt/vol) sodium dodecyl sulfate (SDS), 0.01% (vol/vol) phenylmethylsulfonyl fluoride, 1.0% (vol/vol) ß-mercaptoethanol], and protein concentration was determined by the method of Lowry et al. (56). Proteins were separated by SDS-PAGE using a 4.5% (wt/vol) Laemmli stacking gel (57) and a 10% (wt/vol) Porzio and Pearson resolving gel (58). Proteins were transferred to Schleicher & Schuell Protran nitrocellulose (Intermountain Scientific, Kaysville, UT) at 4 C. Blots were blocked overnight at 4 C in PBS plus 0.1% (vol/vol) Tween 20 containing 5% (wt/vol) nonfat milk. The MUC1 primary antibody 214D4 (kindly provided by Dr. John Hilkens, The Netherlands Cancer Institute, Amsterdam, The Netherlands) (59) was added to a final dilution of 1:10,000 and incubated overnight at 4 C. Blots were incubated for 2 h at 4 C with horseradish peroxidase-conjugated sheep anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) at a final dilution of 1:200,000 in blocking solution. SuperSignal West Dura Extended Duration Substrate (Pierce, Rockford, IL) was used for detection, per the manufacturer’s instructions. Blots were exposed to x-ray film, and signal intensities were quantitated using the Alpha Imager 1D-Multi Function (Alpha Innotech, San Leandro, CA).

Whole-Cell Extract (WCE) Preparation
Insect cell extracts containing PRA or PRB were made as described previously (60). Briefly, Spodoptera frugiperda (Sf9) insect cells were infected with hPR-Ahis or hPR-Bhis recombinant baculovirus at a multiplicity of infection of 1.0 for 48 h at 27 C. R5020 was added 24 h postinfection to a final concentration of 200 nM. Cells were pelleted and washed once with PBS before freezing at –80 C. To prepare WCEs, approximately 6 x 107 cells were lysed in TEDG buffer [10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, 10% (vol/vol) glycerol] plus 0.5 M NaCl and Protease Inhibitor Cocktail Set (PICS) III (Calbiochem, La Jolla, CA). The suspension was cleared by ultracentrifugation at 100,000 x g for 30 min. The supernatant was dialyzed against TEDG overnight at 4 C to reduce the concentration of NaCl. WCE protein content was determined using the BCA Protein Assay kit (Pierce).

EMSAs
EMSA and supershift assays for analysis of PR binding to the MUC1 promoter were carried out using the LightShift Chemiluminescent EMSA kit (Pierce). The rat tyrosine aminotransferase PRE (5'-GATCCTGTACAGGATGTTCTAGCTACA-3') (61) was biotin-labeled using the Biotin 3'-End DNA Labeling kit (Pierce) and used as a probe. The double-stranded competitor oligonucleotides used were as follows: the –570 to –523 region of the MUC1 promoter (570/523MUC; 5'-GG-GGCGGGGACTGTGGGTTCAGGGTAGAACTGCGTGTGGA-ACGGGACAGGGAGCGGTTAGA-3'); mutant consensus PRE (mutPRE; 5'-GATCCTcaACAGGATcaTCTAGCTACA-3', mutant bases in lowercase); the potential PRE from –553 to –535, flanked by additional native sequence (PRE1; 5'-CAGGGTAGAACTGCGTGTGGAACGGGAC-3'); and mutPRE1 (5'-CAGGGTcGgcCgGCGTGTGGAACG-3'). All synthetic oligonucleotides were made by Sigma-Genosys. Binding reactions were carried out essentially as described (62). Briefly, WCE (2.4 µg PRA WCE or 1.5 µg PRB WCE) was incubated at 4 C for 30 min in 1x binding buffer [1 µg poly(dA·dT), 1 µg/µl gelatin, 100 mM Tris (pH 7.5), 500 mM NaCl, 50 mM dithiothreitol, 20 mM MgCl2, and 10% (vol/vol) glycerol], and unlabeled (cold) competitor DNA (20 pmol) was added as indicated. For supershifts, 8 µg anti-PR antibody (Santa Cruz Biotechnology) or immunopurified rabbit IgG (Jackson ImmunoResearch) was added to the solution and incubated at 4 C for 30 min before addition of binding buffer. Biotin-labeled probe was added and the mixture was incubated at 4 C for an additional 1 h. Native 4% (wt/vol) polyacrylamide gels (29:1 polyacrylamide:bisacrylamide ratio) were preelectrophoresed at 200 V for 2 h in 0.5x TBE [45 mM Tris base, 45 mM boric acid, 1 mM EDTA (pH 8.0)] in a water-cooled apparatus. Samples were loaded onto the gels and subjected to electrophoresis at 80 V for 6 h, and then transferred onto an uncharged nylon membrane (Amersham Biosciences, Piscataway, NJ) at 350 mA for 2 h at 4 C. DNA was UV cross-linked to the membrane using a UV Stratalinker 24 (Stratagene), and the biotin-end-labeled target was detected using the Chemiluminescent Nucleic Acid Detection Module (Pierce) per the manufacturer’s instructions.

ChIP Assays
HEC-1A cells were grown in 150-cm3 cell culture flasks until they were 80–90% confluent. Cells were transiently transfected with 30 µg hPRA, 30 µg hPRB, or 15 µg hPRA and 15 µg hPRB using LipofectAMINE 2000 (Invitrogen). The cells then were recovered 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. Chromatin was extracted as described (63) and stored at –80 C. Chromatin was sheared to an average length of 200–500 bp by sonication for three 30-sec pulses on ice. The samples were cleared by centrifugation at 14,000 rpm for 10 min at 4 C, diluted 1:1 with dilution buffer [0.01% (wt/vol) SDS, 1.1% (vol/vol) Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-Cl (pH 8.1), 167 mM NaCl] plus PICS III, and the protein concentration was determined by BCA assay as described above. The chromatin was precleared by addition of normal rabbit serum (Jackson ImmunoResearch) and Protein A/G beads (Santa Cruz Biotechnology, Santa Cruz, CA) that had been blocked with BSA and salmon sperm DNA. Chromatin was divided into samples containing 200 µg total protein and incubated with 2 µg anti-PR antibody C-20 (Santa Cruz Biotechnology) at 4 C overnight. Blocked Protein A/G beads were added, and the samples were incubated overnight at 4 C. The beads were washed twice with dialysis buffer [2 mM EDTA, 50 mM Tris (pH 8.0), 0.2% (wt/vol) Sarkosyl] containing PICS III and six times with immunoprecipitation wash buffer [100 mM Tris-Cl (pH 9.0), 500 mM LiCl, 1% (vol/vol) Nonidet P-40, 1% (wt/vol) deoxycholic acid] plus PICS III, and eluted in 50 mM NaHCO3 and 1% (wt/vol) SDS. RNase A (10 µg) and NaCl (final concentration of 0.3 M) were added, and the samples and inputs (1/4 total input chromatin) were incubated at 65 C overnight to reverse cross-linking. Samples were ethanol precipitated and resuspended in PK buffer [10 mM Tris-Cl (pH 7.6), 5 mM EDTA, 0.24% (wt/vol) SDS] plus 10 µg proteinase K (Promega) and incubated at 45 C overnight. DNA was purified with the QIAquick PCR Purification kit (Qiagen, Valencia, CA). PCR with primers specific to the –618/–500 (forward: 5'-CTTTCTCCAAGGAGGGAACC; reverse: 5'-GGAATAGCCCCACCCTTCTA; 118-bp product) and –163/–23 (forward: 5'-AGCCCTTGTACCCTACCCAG; reverse: 5'-GCTTTATACCGGTCCCCC; 140-bp product) regions of the MUC1 promoter was carried out under the following conditions: 1) 94 C for 2 min, 2) 35 cycles of 94 C for 1 min, 59 C for 1 min, and 72 C for 1 min, and 3) final extension at 72 C for 10 min. PCR products were resolved on a 1.2% (wt/vol) agarose gel containing ethidium bromide and visualized under UV light. Real-time PCR was performed using the QuantiTect SYBR Green PCR kit (Qiagen), using the –618/–500 primers described above, in the iCycler iQ real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA). After 15 min of incubation at 95 C, 40 cycles were performed as follows: denaturation at 95 C for 1 min, annealing at 62.0 C for 1 min, and extension at 72 C for 1 min. Starting concentrations were determined using a standard curve of known amounts of 1.4MUC plasmid and expressed relative to starting input concentrations.

Experimental Animals
Genetically modified mice (C57BL/129SV background) were generated as described previously (22, 64), and were maintained in the animal facility at Baylor College of Medicine (BCM) (Houston, TX). All animal experimentation described was conducted in accordance with accepted standards of humane animal care. Protocols for animal work were approved by the BCM Institutional Committee on Animal Care.

Hormone Treatments and Tissue Collection
Mice (five per group) were ovariectomized at 6–8 wk of age, and 14 d later, animals were given daily sc injections of sesame oil (vehicle control), sesame oil solution of E2 (100 ng), or E2 (100 ng) plus P (1 mg) for 3 d, and uterine tissue was collected on d 4.

Immunohistochemistry
Uterine sections (5 µm) were prepared from tissues fixed in 4% (wt/vol) paraformaldehyde and paraffin-embedded. Deparaffinization, antigen retrieval, and immunofluorescence analysis with the CT-1 antibody were performed as described (65).

RNA Isolation and Real-Time RT-PCR Analysis
Uterine tissue was collected and snap-frozen in liquid N2 for RNA isolation. Total RNA was isolated from pooled tissues (five animals) using Trizol (Invitrogen) according to manufacturer’s protocol, quantified by UV spectrophotometry, and stored in diethylpyrocarbonate water (Ambion, Austin, TX) at –80 C. Total RNA was reversed-transcribed using the GeneAmp RNA PCR kit (Applied Biosystems, Foster City, CA). One microgram of total RNA was reverse-transcribed in a final reaction volume of 20 µl, using random hexamers, for 10 min at 25 C, 15 min at 42 C, 5 min at 99 C, and 5 min at 5 C. Real-time PCR was performed as described above using the following primer sequences: mouse Muc1, 5'-TACCACTCCAGTCCACAGCA and 5'-GTCTTCAGGAGCTCTGGTGG; mouse cytokeratin 18 (CK18), 5'-GGGCTTCATTTGCTGTCTGT and 5'-TAGTCCCAGCATTGGGTAGC; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-GCTGAGTATGTCGTGGAGTC and 5'-TTGGTGGTGCAGGATGC-ATT (66). The GAPDH standard was obtained as described (67). After 15 min of incubation at 95 C, 42 cycles were performed as follows: denaturation at 95 C for 1 min, annealing at 63.8 C (Muc1) or 58 C (CK18 and GAPDH) for 1 min, and extension at 72 C for 1 min. Results are expressed as abundance of Muc1 mRNA relative to K18 mRNA (to control for the amount of epithelial cells in the samples) by the {Delta}{Delta}Ct method (68), normalized to the amount of GAPDH.

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 one-way ANOVA followed by the Tukey-Kramer multiple comparisons test using the GraphPad InStat, version 3.05, software (GraphPad Software, San Diego, CA).


   ACKNOWLEDGMENTS
 
We thank Dr. John Koh for providing the ER-{alpha} and ERE plasmids, Dr. Sandra Gendler for the MUC1 promoter plasmid, Dr. Pierre Chambon for the PRB and PRA expression plasmids, Dr. Shan Lu for the p21-luc plasmid, and Dr. Randal C. Jaffe for the mutant PRB expression plasmid. We thank Dr. John Hilkens for his generous gift of the MUC1 antibody, 214D4. We thank Kurt Christensen and the Baylor College of Medicine Cancer Center Recombinant Protein Expression/Proteomics Core for production of the PRA- and PRB-infected insect cells. We are greatly indebted to Dr. Errin Lagow, Dr. Amantha Thathiah, Ben Rohe, and Neeraja Dharmaraj for many helpful discussions. We are especially grateful to Sharon Kingston and Doreen Anderson for their expert secretarial assistance.


   FOOTNOTES
 
This work was supported by National Institutes of Health (NIH) Grant UO1 HD 29963 (to D.D.C.) as part of the National Cooperative Program on Trophoblast-Maternal Tissue Interactions, NIH Grant HD32007 (to O.M.C.), NIH Grant CA46938 (to D.P.E.), and National Science Foundation Integrative Graduate Education and Research Traineeship Fellowship (Grant 0221651) (to M.J.B.).

M.J.B., B.M.-J., O.M.C., and D.P.E. have nothing to declare. J.J. consulted for Alcon Laboratories, Inc. D.D.C. consulted for and received lecture fees from Alcon Laboratories, Inc.

First Published Online June 1, 2006

Abbreviations: ChIP, Chromatin immunoprecipitation; DBD, DNA binding domain; E2, estrogen; ER, estrogen receptor; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GE, glandular epithelia; KO, knockout; LE, luminal epithelia; P, progesterone; PR, progesterone receptor; PRE, P-responsive element; SDS, sodium dodecyl sulfate; STAT, signal transducer and activator of transcription; WCE, whole-cell extract.

Received for publication August 26, 2005. Accepted for publication May 22, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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