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Molecular Endocrinology, doi:10.1210/me.2003-0105
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Molecular Endocrinology 18 (3): 521-532
Copyright © 2004 by The Endocrine Society

Fos and Jun Inhibit Estrogen-Induced Transcription of the Human Progesterone Receptor Gene through an Activator Protein-1 Site

Larry N. Petz, Yvonne S. Ziegler, Jennifer R. Schultz and Ann M. Nardulli

Department of Molecular and Integrative Physiology, University of Illinois, Urbana, Illinois 61801

Address all correspondence and requests for reprints to: Ann M. Nardulli, Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, Illinois 61801. E-mail: anardull{at}life.uiuc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The progesterone receptor (PR) gene is activated by estrogen in normal reproductive tissues and in MCF-7 human breast cancer cells. Although it is typically thought that estrogen responsiveness is mediated through estrogen response elements (EREs), the human PR gene lacks a palindromic ERE sequence. We have identified an activating protein-1 (AP-1) site at +745 in the human PR gene that bound purified Fos and Jun and formed a complex with Fos/Jun heterodimers present in MCF-7 nuclear extracts. Surprisingly, mutating the +745 AP-1 site in the context of a 1.5-kb region of the PR gene significantly enhanced estrogen receptor (ER) {alpha}-mediated transactivation, suggesting that the wild-type +745 AP-1 site plays a role in inhibiting PR gene expression in the presence of hormone. In support of this idea, transient transfection assays demonstrated that increasing levels of Fos and Jun repressed transcription of a reporter plasmid containing the +745 AP-1 site. Fos levels were transiently increased, ER{alpha} levels were decreased, and Jun was dephosphorylated after MCF-7 cells were treated with estrogen. Chromatin immunoprecipitation assays demonstrated that Jun was associated with the +745 AP-1 site in the endogenous PR gene in the presence and in the absence of estrogen, but that ER{alpha} and Fos were only associated with the +745 AP-1 site after estrogen treatment of MCF-7 cells. Our studies suggest that the human PR gene is regulated by multiple transcription factors and that the differential binding of these dynamically regulated trans-acting factors influences gene expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE HORMONE 17ß-ESTRADIOL (E2) is critical for the development and maintenance of reproductive tissues (1, 2). The effects of estrogen are mediated through a ligand-activated transcription factor, the estrogen receptor (ER). The interaction of the E2-occupied ER{alpha} with EREs in target genes is considered the initiating event in transcription activation (3, 4). In addition to its interaction with EREs, ER{alpha} can also interact with DNA-bound transcription factors to modulate gene expression (reviewed in Refs. 5 and 6). ER{alpha} confers estrogen responsiveness to a number of genes in this manner including the human progesterone receptor (PR) gene by interacting with DNA-bound AP-1 and Sp1 proteins (7, 8, 9).

Previous studies have demonstrated that maximal PR mRNA and protein levels are reached after MCF-7 human breast cancer cells have been exposed to E2 for 3 d (10, 11, 12). Two discrete promoters, A (+464 to +1105) and B (-711 to +31), are thought to be responsible for the production of the 94-kDa PR-A and the 120-kDa PR-B proteins, respectively (13). Interestingly, despite the fact that both promoters are estrogen responsive (13, 14), neither contains a palindromic ERE sequence.

In this study, we have identified an AP-1 site from +745 to + 751 in the human PR gene that bound Fos and Jun in vitro and was associated with Jun in the endogenous PR gene both in the presence and absence of E2 but was associated with ER{alpha} and Fos only after E2 treatment of MCF-7 cells. Our studies suggest that Fos, Jun, and ER{alpha} associate with the +745 AP-1 site in the human PR gene and assist in regulating expression of the PR gene.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The +745 AP-1 Site Is Conserved in the Human, Rabbit, Mouse, and Rat PR Genes
A previous study of the rabbit PR gene reported that an imperfect ERE (+706/+718), which overlaps with the PR-B translation start site, was implicated in estrogen-responsive gene expression (15). Examination of the corresponding region of the human PR gene indicated that this ERE was not conserved (13). However, a putative AP-1 site (TGACTGA) from +745 to +751, hereafter referred to as the +745 AP-1 site, was present and differed from the consensus AP-1 site (TGAG/CTCA; see Refs. 16 and 17) by a single base pair. Interestingly, unlike the imperfect ERE found in the rabbit gene, the +745 AP-1 site is completely conserved in human, rabbit, mouse, and rat PR genes (Fig. 1Go).



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  		Fig. 1. Sequence of PR Gene Sequence

The sequence of the +745 AP-1 site in the human PR gene is shown in bold. The corresponding sequences of the rabbit, mouse, and rat PR genes and the numbers of the nucleotide sequence for each species are also included. The region of the rabbit PR gene containing an imperfect palindromic ERE, previously described by Savouret et al. (51 ), is underlined.

 
The +745 AP-1 Site Is Involved in Estrogen-Mediated Transcription of the PR Gene
To determine whether the putative +745 AP-1 site could confer estrogen responsiveness to a simple promoter, chloramphenicol acetyl transferase (CAT) reporter plasmids containing a TATA sequence alone (TATA-CAT) or the +745 AP-1 site and a TATA box (+745 AP-1 TATA-CAT) were tested for their abilities to activate transcription. U2 osteosarcoma cells were transfected with a CAT reporter plasmid and a ß-galactosidase expression vector in the absence (-ER{alpha}) or in the presence (+ER{alpha}) of a human ER{alpha} expression vector. When the reporter plasmid containing the +745 AP-1 site and the ER{alpha} expression vector were used, exposure of the transfected cells to E2 resulted in a 2.7-fold increase in CAT activity (Fig. 2Go, +745 AP-1 TATA-CAT). In contrast, hormone treatment did not affect CAT activity when the reporter plasmid contained the TATA sequence alone (TATA-CAT). When the ER{alpha} expression vector was not included, no difference in CAT activity was observed regardless of hormone treatment indicating that the estrogen-induced increase in CAT activity was dependent on the presence of ER{alpha} and E2. These experiments directly demonstrated that ER{alpha} is required for estrogen responsiveness and that the +745 AP-1 site can confer estrogen responsiveness to a heterologous promoter and may assist in mediating estrogen’s effects on the endogenous PR gene. Although we would have preferred to use MCF-7 cells for our transfection experiments, the low transfection efficiency of these cells and the limited activity of the +745 AP-1 site prevented us from doing so. U2 osteosarcoma (U2-OS) cells do, however, provide an appropriate model cell line to examine PR gene expression because transient transfection of these cells with an ER{alpha} expression vector activates transcription of the endogenous PR gene (data not shown). Thus, U2-OS cells have the proteins required for regulating PR gene expression and responding to hormone treatment.



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  		Fig. 2. Transient Cotransfections with Reporter Plasmids Containing or Lacking the +745 AP-1 Site

U2-OS cells were transfected with a reporter plasmid containing the +745 AP-1 site and a TATA box (+745 AP-1 TATA-CAT) or the TATA box alone (TATA-CAT) and a ß-galactosidase expression plasmid. A human ER{alpha} expression plasmid was not (-ER{alpha}) or was (+ER{alpha}) included as indicated. Cells were treated with ethanol vehicle or 10 nM E2 for 24 h and CAT activity was determined. Data from three independent experiments were combined and values are presented as the mean ± SEM. Student’s t tests demonstrated that the E2-treated samples were statistically different from the corresponding ethanol-treated samples (P < 10-4) when the +745 AP-1 TATA-CAT and a human ER{alpha} expression vector were used.

 
Proteins Present in Nuclear Extracts from E2-Treated MCF-7 Cells Bind to the AP-1 Site
To determine whether proteins present in MCF-7 cells might help mediate estrogen’s effects on PR gene expression by interacting with the +745 AP-1 site, gel mobility shift assays were carried out. 32P-labeled oligos containing the +745 AP-1 site were incubated with nuclear extracts from MCF-7 cells that had been treated with E2 for 0, 2, 24, or 72 h and fractionated on a nondenaturing acrylamide gel. Interestingly, a distinct protein-DNA complex was formed with nuclear extracts from MCF-7 cells that had been treated with E2 for 72 h (Fig. 3AGo, lane 5, <- Fos /Jun), but not with extracts from cells that had been treated with E2 for 0, 2, or 24 h (lanes 2–4).



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  		Fig. 3. Gel Mobility Shift Assays with an AP-1-Containing Oligo and MCF-7 Nuclear Proteins

32P-labeled oligos containing the +745 AP-1 site were incubated without (lanes 1) or with 20 µg of nuclear extract from 0, 2, 24, or 72 h E2-treated MCF-7 cells (panel A, lanes 2–5) or MCF-7 cells that had been treated with E2 for 72 h (panel B, lanes 2–5). Antibodies to c-Fos, c-Jun, or ER{alpha} were added to the binding reactions as indicated. The 32P-labeled oligos were fractionated on a nondenaturing gel and visualized by autoradiography. Complexes containing Fos/Jun are indicated to the right of the figure (<-Fos/Jun).

 
To determine which AP-1 proteins were involved in formation of the protein-DNA complex, 32P-labeled oligos containing the +745 AP-1 site were combined with nuclear extracts from MCF-7 cells that had been treated with E2 for 72 h and fractionated on an acrylamide gel. Proteins present in the nuclear extracts bound to the +745 AP-1 site (Fig. 3BGo, lane 2, <- Fos/Jun). One of the protein-DNA complexes was disrupted by the inclusion of an antibody that recognizes c-Fos but does not cross-react with Fos B, Fra-1, or Fra-2 (lane 3). Addition of an antibody that recognizes c-Jun, but does not cross-react with Jun B or Jun D, also disrupted the protein-DNA complex (lane 4). These data indicate that the endogenously expressed c-Fos and c-Jun present in MCF-7 nuclear extracts bind to the +745 AP-1 site. The abilities of both Fos and Jun antibodies to disrupt the protein-DNA complex suggest that Fos and Jun bind to the AP-1 site as a heterodimer. In contrast to these findings, the ER{alpha}-specific antibody H151 (lane 5) did not affect the protein-DNA complex. Our inability to detect ER{alpha} binding to the +745 AP-1 site was probably due to the low level of receptor in nuclear exacts, which we estimated was approximately 100-fold lower in our gel shift binding reactions than is present in MCF-7 nuclei (7).

Fos Levels Are Increased, ER{alpha} Levels Are Decreased, and Jun Is Dephosphorylated after E2 Treatment of MCF-7 Cells
The failure of Fos and Jun to bind the +745 AP-1 site when MCF-7 cells had been exposed to estrogen for shorter time periods (Fig. 3AGo) prompted us to determine whether hormone exposure might influence expression of Fos and/or Jun proteins in MCF-7 cells. Western blot analysis was performed with nuclear extracts from MCF-7 cells that had been treated with E2 for 0, 2, 24, or 72 h. The c-Fos-specific antibody detected two proteins with apparent molecular masses of 55 and 61 kDa (Fig. 4AGo, Fos ->). The c-Jun-specific antibody detected two proteins with apparent molecular masses of 39 and 42 kDa (Jun ->). Because both Fos and Jun can be phosphorylated (18, 19, 20, 21), the presence of two bands for each of the proteins most likely represented different phosphorylation states of these AP-1 proteins.



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  		Fig. 4. Western Blot Analysis of Nuclear Extracts from E2-Treated MCF-7 Cells

Twenty micrograms of nuclear extract from MCF-7 cells that had been treated with E2 for the indicated times were fractionated on a denaturing gel, transferred to a nitrocellulose membrane, and incubated with a c-Fos-specific antibody, an antibody that recognizes phosphorylated and dephosphorylated c-Jun (panel A), an antibody that recognizes phosphoylated, but not dephosphorylated c-Jun (panel B), or an antibody that recognizes ER{alpha} (panel C). A chemiluminescent system was used to detect the proteins.

 
Although Fos levels transiently increased after 2 h of E2 treatment, the levels of Jun did not change substantially with E2 treatment. There was, however, an increase in the more rapidly migrating form of Jun and a concomitant decrease in the more slowly migrating form of Jun with increasing time of E2 treatment (Fig. 4AGo). We felt that the more rapidly and more slowly migrating forms were most likely dephosphorylated and phosphorylated Jun, respectively. To verify that Jun was dephosphorylated after exposure of MCF-7 cells to E2, Western blot analysis was performed with nuclear extracts from MCF-7 cells that had been treated with E2 for 0, 24, or 72 h, and the blot was probed with an antibody that recognizes only phosphorylated Jun. As seen in Fig. 4BGo, decreased levels of phosphorylated Jun were detected after increasing times of E2 treatment (Jun ->). Thus, both Fos and Jun were affected by E2 treatment. Fos was transiently increased and Jun was dephosphorylated after E2 treatment of MCF-7 cells.

The expression of ER{alpha} protein was also monitored after MCF-7 cells had been treated with E2 (Fig. 4CGo). ER{alpha} levels diminished significantly after MCF-7 cells had been treated with E2 for increasing periods of time.

Purified Fos and Jun Bind to an Oligo Containing the +745 AP-1 Site
Our gel mobility shift assays indicated that Fos and Jun present in MCF-7 nuclear extracts could bind to the +745 AP-1 site. To determine whether binding of Fos and Jun required other nuclear proteins, gel mobility shift experiments were carried out with purified Fos and Jun. 32P-labeled oligos containing the +745 AP-1 site were incubated with increasing amounts of purified Fos and Jun and fractionated on a nondenaturing acrylamide gel. At the lowest concentration of Fos and Jun used (5 nM), a faint gel-shifted band was visible (Fig. 5Go, lane 2, <- Fos /Jun). The gel-shifted band intensified as increasing concentrations of Fos and Jun were added to the binding reaction (lanes 3–6). The inclusion of the c-Fos-specific (lane 7) or c-Jun-specific antibody (lane 8) supershifted the protein-DNA complex. These findings again support the idea that Fos and Jun formed a stable heterodimeric complex with the +745 AP-1 site. In contrast to these findings, neither Fos nor Jun alone was able to bind to the + 745 AP-1 site (data not shown).



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  		Fig. 5. Gel Mobility Shift Assays with an AP-1-Containing Oligos and Purified Fos and Jun

32P-labeled oligos containing the +745 AP-1 site were incubated alone (lane 1) or with 5, 10, 20, 50 (lanes 2–5) or 70 nM (lanes 6–8) purified Fos and Jun. c-Fos- or c-Jun-specific antibody (Ab) was added to the binding reaction as indicated. Complexes containing Fos and Jun are indicated to the right of the figure (<-Fos/Jun).

 
ER{alpha} Enhances the Binding of Fos and Jun to the +745 AP-1 Site
We recently demonstrated that ER{alpha} enhanced binding of Fos and Jun to an AP-1 site in another region (+90 to +96) of the human PR gene (8). This +90 AP-1 site contains a DNA sequence (TGAGTGA) that is nearly identical with the +745 AP-1 site (TGACTGA), and each differs from the consensus AP-1 site (TGAG/CTCA; see Refs. 16 and 17) by only 1 bp. To determine whether baculovirus-expressed, purified ER{alpha} might influence binding of Fos and Jun to the +745 AP-1 site, gel mobility shift assays were carried out. When 32P-labeled oligos containing the +745 AP-1 site were incubated with 12.5 nM purified Fos and Jun and fractionated on a nondenaturing acrylamide gel, a single faint Fos/Jun complex was formed (Fig. 6Go, Fos/Jun ->, lane 1). Addition of increasing amounts of purified E2-occupied ER{alpha} to the binding reaction resulted in greatly enhanced Fos/Jun binding and in the formation of a second, faster migrating protein-DNA complex (lanes 2–4). To determine whether the more rapidly migrating protein-DNA complex contained ER{alpha}, additional gel mobility shift assays were carried out. When 32P-labeled oligos containing the +745 AP-1 site were coincubated with purified Fos, Jun, and ER{alpha} and fractionated on a nondenaturing acrylamide gel, two gel-shifted bands were formed (Fig. 6Go, lane 5). The inclusion of c-Fos-specific antibody (lane 6) or c-Jun-specific antibody (lane 7) supershifted the slower migrating protein-DNA complex. Addition of an ER{alpha}-specific antibody (lane 8) supershifted the more rapidly migrating protein-DNA complex. These data indicated that the upper gel-shifted band corresponded to the Fos/Jun-DNA complex (Fos/Jun ->), whereas the lower gel shifted band corresponded to the ER{alpha}-DNA complex (ER{alpha} ->). Thus, both ER{alpha} and Fos/Jun bound to the +745 AP-1 site and ER{alpha} significantly enhanced Fos/Jun binding. However, ER{alpha} and Fos/Jun did not form a stable trimeric protein-DNA complex, but rather bound to the +745 AP-1 site independently.



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  		Fig. 6. Gel Mobility Shift Assays with an AP-1-Containing Oligos and Purified Fos, Jun, and ER{alpha}

32P-labeled oligos containing the +745 AP-1 site were incubated with 12.5 nM purified Fos and Jun (lanes 1–8) and 100 (lane 2), 200 (lane 3), or 400 (lanes 4–8) fmol purified ER{alpha}. c-Fos-, c-Jun-, or ER{alpha}-specific antibody (Ab) was added to the binding reactions as indicated. Complexes containing Fos/Jun or ER{alpha} are indicated to the left of the figure.

 
Mutating the +745 AP-1 Site in the Context of the PR Promoter Increases Transcription
The +745 AP-1 site was capable of conferring estrogen responsiveness to a heterologous promoter (Fig. 1Go), but we felt it was important to assess the ability of this site to influence transcription in the context of the PR gene sequence. When a 1.5-kb region of the PR gene (-711 to +817) was included in the TATA-CAT reporter plasmid, transcription was increased 28-fold in the presence of E2 (Fig. 7Go, -711/+817 TATA-CAT, +ER{alpha}). Surprisingly, when 2 bp in the +745 AP-1 site were mutated from TGACTGA to GCGAGTGA (-711/+817 mut 1 +745 TATA-CAT, +ER{alpha}), transcription was significantly enhanced (33-fold) in the presence of E2. Because these results were unexpected, a second reporter plasmid, which contained different mutations in the +745 AP-1 site (CGACCTG) was constructed. When this reporter plasmid was used in transient transfection assays, transcription was increased 37-fold in the presence of hormone (-711/+817 mut 2 +745 TATA-CAT, +ER). These results were particularly surprising in light of the fact that the identical mutations in the +90 AP-1 site in this 1.5-kb region of the PR gene dramatically decreased E2-mediated activation (8). The increases in transcription observed were E2 and ER{alpha} dependent because no change in transcription was observed when the ER{alpha} expression vector was omitted (-ER{alpha}). Our findings suggest that, rather than increasing transcription, the +745 AP-1 site may actually play a role in tempering PR gene expression in the presence of hormone.



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  		Fig. 7. Transient Cotransfections with Reporter Plasmids Containing the 1.5-kb Region of the PR Gene with a Wild-Type or Mutant +745 AP-1 Site

U2-OS cells were transfected with a reporter plasmid containing a 1.5-kb region (-711 to +817) of the PR gene with wild type nucleotide sequence (-711/+817 TATA-CAT) or with two (-711/+817 mut 1 +745 TATA-CAT) or with 4-bp (-711/+817 mut 2 +745 TATA-CAT) mutations in the +745 AP-1 site and a ß-galactosidase expression plasmid. A human ER{alpha} expression plasmid was not (-ER{alpha}) or was (+ER{alpha}) included as indicated. Cells were treated with ethanol vehicle or 10 nM E2 for 24 h, and CAT activity was determined. Data from three independent experiments were combined, and values are presented as the mean ± SEM. ANOVA was used to determine that each E2-treated sample was statistically different from the other E2-treated samples when the ER{alpha} expression vector was included (P < 0.001).

 
Fos, Jun, and ER{alpha} Selectively Interact with the Wild-Type and Mutant +745 AP-1 Containing Oligos
To try to understand how mutating the +745 AP-1 site could increase transcription, we examined the abilities of Fos, Jun, and ER{alpha} to bind to the wild-type and mutant +745 AP-1 sites in gel mobility shift assays. 32P-labeled oligos containing the wild-type or a mutated +745 AP-1 site were incubated with purified Fos, Jun, or ER{alpha} and fractionated on a nondenaturing acrylamide gel. When Fos and Jun were incubated with the wild-type +745 AP-1 site, a single protein-DNA complex containing Fos/Jun was observed (Fig. 8Go, wt +745, lane 1, Fos/Jun ->). Similarly, when purified ER{alpha} was incubated with the wt +745 AP-1 site, a single protein-DNA complex containing ER{alpha} was observed (lane 2, ER{alpha} ->). Coincubation of Fos, Jun, and ER{alpha} resulted in the appearance of both the Fos/Jun- and ER{alpha}-DNA complexes (lane 3). In contrast, when mut 1 oligos containing 2-bp mutations in the +745 AP-1 site were incubated with Fos, Jun, and/or ER{alpha}, no gel shifted bands were observed (mut 1 +745, lanes 4–6). Interestingly, when oligos containing 4-bp mutations in the +745 AP-1 site were incubated with purified Fos and Jun, the intensity of the gel shifted band was sharply decreased (mut 2 +745, lane 7) compared with wt +745 AP-1 Fos/Jun binding. ER{alpha} alone bound efficiently to the mutant oligo (lane 8). Surprisingly, incubating the mut 2 +745 oligos with ER{alpha}, Fos, and Jun resulted in diminished Fos/Jun binding (compare lanes 9 and 7). It should be noted that the molar ratios of Fos/Jun and ER{alpha} were held constant and the binding reactions were processed in parallel and run on the same gel. Thus the differences observed in binding of Fos/Jun and ER{alpha} to these sites reflected differences in protein-DNA interactions and could not be attributed to differences in experimental conditions.



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  		Fig. 8. Gel Mobility Shift Assays with Wild-Type (wt) or Mutant AP-1-Containing Oligos and Purified Fos, Jun, and ER{alpha}

32P-labeled oligos containing the +745 AP-1 site with wt nucleotide sequence (wt +745, lanes 1–3) or with 2-bp (mut 1 +745, lanes 4–6 and mut 1L +745, lanes 10–12) or 4-bp (mut 2 +745, lanes 7–9 and 13–15) mutations were incubated with 12.5 nM purified Fos and Jun and/or with 400 fmol purified ER{alpha} as indicated. Complexes containing Fos and Jun or ER{alpha} are indicated to the left of the figure. The mut 1L +745 oligos were identical with the mut 1 +745 oligos, except that they were extended by two and five additional nucleotides at their 5' and 3' ends, respectively. The autoradiogram to the right was exposed to film for a more extended time to try to visualize any protein binding.

 
Because the mut 1 +745 AP-1 oligos were somewhat shorter than the other oligo pairs and might limit protein binding, we also conducted gel mobility shift experiments with longer oligos, which had nucleotide sequence identical with the mut 1 +745 AP-1 oligos but were extended at their 5' and 3' ends by two and five additional nucleotides, respectively (mut 1L +745 oligos). Even when the gel was exposed to film for a more extended period of time, we were still unable to detect any Fos/Jun- or ER{alpha}-DNA complexes with this second set of oligos. These combined results suggest that the higher transcriptional activity observed with the mutant +745 AP-1 sites is most likely due to decreased Fos and Jun binding and, in the case of second mutation, enhanced ER{alpha} binding. Inherent in these findings is the suggestion that Fos and Jun may act at the +745 AP-1 site to limit PR gene expression.

Fos and Jun Inhibit Transcriptional Activity of the +745 AP-1 Site
To determine whether Fos and Jun could repress transcription through the +745 AP-1 site, U2-OS cells were transfected with a CAT reporter plasmid containing the wild-type +745 AP-1 site (+745 AP-1 TATA-CAT), a ß-galactosidase expression vector, and a human ER{alpha} expression vector. Exposure of the transfected cells to E2 resulted in a 2.9-fold increase in CAT activity (Fig. 9Go, +745 AP-1 TATA-CAT). The inclusion of c-Fos and c-Jun expression vectors resulted in a significant decrease in transcription in the presence of E2. At the highest concentrations of c-Fos and c-Jun expression vectors used (250 ng), transcription was decreased to 1.2-fold. In contrast to these findings, a reporter plasmid containing the +90 AP-1 site was not affected by increased expression of Fos and Jun (data not shown). These data suggest that Fos and Jun do function as transcriptional repressors at the +745 AP-1 site.



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  		Fig. 9. Transient Cotransfections with Reporter Plasmids Containing the +745 AP-1 Site

U2-OS cells were transfected with a reporter plasmid containing the +745 AP-1 site and a TATA box (+745 AP-1 TATA-CAT), a ß-galactosidase expression plasmid, and a human ER{alpha} expression plasmid. c-Fos and c-Jun expression vectors were included as indicated. Cells were treated with ethanol vehicle or 10 nM E2 for 24 h and CAT activity was determined. Data from two independent experiments were combined (n = 8), and values are presented as the mean ± SEM. ANOVA was used to determine that inclusion of the Fos and Jun expression vectors resulted in dose-dependent decreases in transcription (P < 0.01 and 0.001).

 
ER{alpha}, Fos, and Jun Are Present at the +745 AP-1 Site in Native Chromatin
Our transient transfection and gel mobility shift assays demonstrated that Fos, Jun, and ER{alpha} bound to the +745 AP-1 site and that E2 and ER{alpha} played a role in regulating transcriptional activity. However, a question of even greater importance is whether these proteins play a role in regulating transcription of the endogenous PR gene in cells that respond to E2 with increased PR gene expression. To determine whether Fos, Jun, or ER{alpha} associate with this region of the endogenous PR gene, chromatin immunoprecipitation (ChIP) assays were carried out with MCF-7 cells that had been treated with ethanol vehicle or E2. A discrete amplified product was obtained when MCF-7 cells had been treated with E2 for 24 or 72 h and the protein-DNA complexes were immunoprecipitated with an ER{alpha}, Fos-, or Jun-specific antibody (Fig. 10Go, +745 AP-1, lanes 8, 9, 11, 12, 14, and 15). Interestingly, an amplified product was also obtained when cells were treated with ethanol and the protein-DNA complexes were immunoprecipitated with a Jun-specific antibody (+745 AP-1, lane 13), indicating that Jun was associated with this region of the PR gene both in the absence and presence of estrogen. In contrast, no amplified product was obtained when cells were treated with ethanol vehicle and the protein-DNA complexes were immunoprecipitated with an ER{alpha}- or Fos-specific antibody (+745 AP-1, lanes 7 and 10). Genomic DNA that had not been subjected to immunoprecipitation was readily amplified (+745 AP-1, input, lanes 1–3), but no amplified product was present when antibody was omitted (-Ab) regardless of hormone exposure (+745 AP-1, lanes 4–6). To ensure that the ER{alpha}-, Fos-, or Jun-containing protein-DNA complexes were specifically immunoprecipitated, we determined whether an upstream region of the PR gene that did not contain an ERE or an AP-1 site could be amplified. No DNA product was observed when an upstream region of the PR gene was examined with an ER{alpha}-, Fos-, or Jun-specific antibody (Control, lanes 7–15) or when no antibody (-Ab) was used for immunoprecipitation regardless of hormone exposure (Control, lanes 4–6). In contrast, when genomic DNA was used, an amplified product was obtained (Control, Input, lanes 1–3). These data demonstrate that Jun is associated with the region of the endogenous PR gene containing the +745 AP-1 site both in the absence and in the presence of E2 but that ER{alpha} and Fos are associated with this site only after E2 treatment of MCF-7 cells.



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  		Fig. 10. ChIP of ER{alpha}-Associated DNA

MCF-7 cells, which had been exposed to ethanol vehicle (-E2) or 10 nM E2 (+E2) for 24 or 72 h, were treated with formaldehyde to form protein-protein and protein-DNA cross-links and sonicated. Immunoprecipitation of the protein-DNA complexes was carried out in the absence (-Ab) or in the presence of ER{alpha}-, c-Fos-, or c-Jun-specific antibody (Ab). Primers flanking the +745 AP-1 site (+745 AP-1) or a region of the PR gene that did not contain an AP-1 site (Control) were used in PCR amplification. Amplified DNA was fractionated on an agarose gel and visualized after ethidium bromide staining. Sheared genomic DNA was used as a positive control (Input).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The PR gene contains a number of putative transcription factor binding sites including an ERE half-site, a CAAT sequence, and Sp1 and AP-1 sites, the sequence and location of which are highly conserved. The +745 AP-1 site residing in the human PR gene is identical in nucleotide sequence to the sequence found in the corresponding region of the rabbit PR gene. Interestingly, the AP-1 site in the rabbit PR gene overlaps with an imperfect, palindromic ERE that has been implicated in regulating PR gene expression (15). Although this imperfect ERE found in the rabbit PR gene is not conserved in the human PR gene, the location and sequence of this AP-1 site is completely conserved in the human, rabbit, rat, and mouse PR genes (13, 22, 23, 24). This high degree of sequence conservation suggests that the +745 AP-1 site may play an important role in regulating expression of the PR gene. Our studies provide evidence that the +745 AP-1 site and its associated trans-acting factors are important in regulating PR gene.

Role of AP-1 Proteins in Regulating PR Gene Expression
AP-1 proteins are comprised of a number of polypeptides including Fos and Jun (20, 25) that dimerize to form complexes at AP-1 sites in target genes (26, 27, 28). Exposure of MCF-7 cells to E2 caused a transient increase in Fos protein levels. These data are consistent with the transient increase in Fos mRNA levels that have been reported after a 1-h treatment of MCF-7 cells with E2 (29). These increases in Fos mRNA and protein levels may be mediated by interaction of the E2-occupied receptor with an ERE in the 5' flanking region of the human fos gene (30), thereby leading to increased gene expression. The transient increase in Fos levels after hormone treatment would presumably increase Fos/Jun heterodimer formation. Our combined in vitro binding and ChIP analyses suggest that Jun/Jun homodimers are associated with the +745 AP-1 site in the absence of hormone and that Fos/Jun heterodimers are associated with this region of the PR gene after hormone treatment.

Although the level of Jun was not substantially altered by E2 treatment, there was a decrease in the level of phosphorylated Jun and a concomitant increase in the level of dephosphorylated Jun in MCF-7 cells with increasing times of E2 treatment. Because phosphorylation of Jun decreases (18, 31) and site-specific dephosphorylation of Jun increases AP-1 binding and activity (18), dephosphorylation of Jun could play a role in enhancing Jun binding and mediating estrogen’s effects on PR gene expression in MCF-7 cells. The decreased binding of phosphorylated Jun may in part explain our inability to detect Fos/Jun binding in gel mobility shift assays when MCF-7 cells had been treated with E2 for 2 or 24 h. However, it is clear from our ChIP assays that Jun was associated with the +745 AP-1 site in native chromatin both in the absence and in the presence of hormone. Although we did not see an increase in the association of Jun with the +745 AP-1 site in our ChIP analysis, the presence of Fos at this site only after hormone treatment would most likely be due to the formation and binding of Fos/Jun heterodimers to this region of the PR gene. Thus, rather than the association of Jun/Jun homodimers with this region as we believe occurs in the absence of hormone, Fos/Jun heterodimers may be the predominant AP-1 proteins present after hormone treatment. Our studies support the idea that there is a dynamic change in the association of AP-1 proteins with the +745 AP-1 site after hormone exposure.

Jun has been implicated in inhibiting differentiation of precursor cells to osteoclasts through an AP-1-mediated pathway (32). This could require the combined effects of a Jun-specific kinase and phosphatase. In fact, it has been suggested that the latent, constitutively phosphorylated Jun present in unstimulated cells is activated by a phosphatase to the active, dephosphorylated form (18, 33).

Role of ER{alpha} in Regulating PR Gene Expression
Estrogen regulates transcription of a number of genes through AP-1 sites including the ovalbumin, c-fos, collagenase, and IGF genes (30, 34, 35, 36). We have demonstrated that ER{alpha} bound directly to the +745 AP-1 site and enhanced Fos/Jun binding to this site. Furthermore, transcription of a promoter containing the +745 AP-1 site required both hormone and the receptor for estrogen responsiveness. More importantly, E2 treatment of MCF-7 cells, which is required for induction of the PR gene, promoted the recruitment of ER{alpha} to the +745 AP-1 site in the endogenous PR gene. The interaction of ER{alpha} with the +745 AP-1 site in vivo may be due to binding of the receptor to DNA, to DNA-bound Jun, or to other coregulatory proteins (37, 38, 39).

A close examination of protein binding to the +745 AP-1 site reveals that either Fos/Jun or ER{alpha} can bind directly to this site in vitro. The AP-1 proteins bind to the AP-1 site (TGACTGA), and ER{alpha} binds to the imperfect ERE half-site (TGACT) within the AP-1 site. When 2 bp of the AP-1 site were mutated (GGAGTGA, mut 1), Fos/Jun binding was disrupted and ER{alpha} binding was also lost because the imperfect ERE half-site was also mutated (GGAGT). Surprisingly, when a reporter plasmid containing 1.5 kb of the PR gene with this mutant AP-1 site was used in transfection assays, transcription was increased suggesting that the wild-type AP-1 site plays a role in limiting PR gene expression. When the +745 AP-1 site was mutated so that ER{alpha} binding was retained, but Fos/Jun binding was decreased (mut 2), transcription was again incrementally increased. These findings support the idea that binding of ER{alpha} and Fos/Jun to the +745 AP-1 site may have opposing effects with ER{alpha} enhancing and Fos/Jun limiting transcription. This raises the intriguing possibility that PR gene expression may vary with changes in the expression and/or status of Fos, Jun, and ER{alpha}. It seems possible that as the levels and/or phosphorylation state of Fos, Jun, and ER{alpha} change after various times of hormone treatment, there would be differences in the interaction of these proteins with the +745 AP-1 site. ER{alpha} binding might be more important in the initial stages of hormone exposure when ER{alpha} levels are high. Then as E2-mediated decreases in ER{alpha} levels occur, enhanced binding of Fos and Jun to these sites may help to restrict ER{alpha} binding and PR gene expression. Likewise, modulation of Fos levels and changes in the phosphorylation state of Jun could also help to influence PR gene expression and foster the formation of a different population of transcription factors with the +745 AP-1 site. It is important to remember, however, that the +745 AP-1 site is only one of a number of cis elements regulating PR transcription. Although the +745 AP-1 site influences transcription, it is not the sole determinant of PR gene expression.

Role of AP-1 Sites in Regulating PR Gene Expression
We have now identified two AP-1 sites in the PR gene that are involved in regulating PR gene expression: the +745 AP-1 site and the +90 AP-1 site (8). The nucleotide sequences of the +745 (TGACTGA) and +90 (TGAGTGA) AP-1 sites are nearly identical and, as might be expected from this similarity in nucleotide sequence, there were similarities in the interaction of ER{alpha} and AP-1 proteins with these two sites. ER{alpha} bound to and enhanced Fos and Jun binding at both AP-1 sites in in vitro binding assays, and both AP-1 sites were effective in conferring estrogen responsiveness to a simple heterologous promoter (8). However, it is also clear that the +90 and +745 AP-1 sites have distinctly different functions in regulating PR gene expression. When the +90 AP-1 site was mutated in the context of a 1.5-kb region of the PR gene, transcription was dramatically reduced (8). Paradoxically, when identical mutations were made in the +745 AP-1 site in the context of the 1.5-kb PR region, transcription was significantly increased. The increase in transcription observed with the mutated +745 AP-1 sites is most likely due to the decreased binding of Fos and Jun to this site. In support of this idea, transcription was significantly decreased when Fos and Jun expression were increased. Thus, it appears that the role of Fos/Jun at the +745 AP-1 site is to limit PR gene expression. Savouret et al. (15) have also demonstrated that either Fos or Jun can inhibit transcription through the corresponding region of the rabbit PR gene.

Distinct differences were also noted in the interaction of Jun with the endogenous +90 and +745 AP-1 sites. Although Jun was associated with the endogenous +745 AP-1 site in the absence and in the presence of hormone, it was associated with the +90 AP-1 site only in the presence of hormone (8). It seems possible that binding of Jun/Jun homodimers to the +745 AP-1 site could help to sustain the low, basal levels of PR protein that are present in MCF-7 cells in the absence of hormone (10). The differences in the abilities of Fos and Jun proteins to interact with the +90 and +745 AP-1 sites in the endogenous PR gene suggest that AP-1 sites may be either stimulatory or inhibitory and that the AP-1 DNA sequence and its flanking nucleotide sequence play important roles in regulating transcription. The importance of DNA sequence flanking AP-1 sites has been suggested in previous studies with the ovalbumin gene (34).

The PR gene represents a particularly interesting model in which to define mechanisms regulating estrogen-responsive gene expression. The contributions of multiple cis elements, the dynamic regulation of numerous trans-acting factors, and the abilities of nearly identical cis elements to have distinct effects on transcription provides target cells with substantial flexibility in responding to environmental and cellular cues and allows fine-tuned control of PR gene expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Oligonucleotides and Plasmid Construction
Oligos containing the +745 AP-1 site (wt +745: 5'-GATCTTCCAGTCGTCATGACTGAGCTGAAGGCAAA-3' and 5'-GATCTTTGCCTTCAGCTCAGTCATGACGACTGGAA-3') or containing mutations in the +745 AP-1 site (mut 1 +745: 5'-GTCCAGTCGTCAGGAGTGAGCTGAAGGC-3' and 5'-GCCTTCAGCTCACTCCTGACGACTGGAC-3', mut 1L +745: 5'-GAGTCCAGTCGTCAGGAGTGAGCTGAAGGCAAAGG-3' and 5'-CCTTTGCCTTCAGCTCACTCCTGACGACTGGACTC- 3, which were identical with the mut 1 +745 oligos except that they contained two and five additional nucleotides at the 5' and 3' ends, respectively, and mut 2 +745: 5'-GAGTCCAGTCGTCACGACCTGGCTGAAGGCAAAGG-3' and 5'-CCTTTGCCTTCAGCCAGGTCGTGACGACTGGACTC- 3') site were annealed and used in gel mobility shift assays or inserted into Bgl II-cut, dephosphorylated chloramphenicol acetyl transferase (CAT) reporter plasmid, TATA-CAT (40), to create +745 AP-1 TATA-CAT containing two copies of the AP-1 site. The construction of -711/+817 TATA-CAT has been previously described (8). The -711/+817 TATA-CAT reporter plasmid was used as a template with oligos (mut 1 +745 or mut 2 +745) containing mutations in the +745 AP-1 site to produce reporter plasmids containing mutations in the +745 AP-1 site (-711/+817 mut 1 +745 TATA-CAT and -711/+817 mut 2 +745 TATA-CAT, respectively) using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The c-Fos and c-Jun expression vectors have been previously described (kindly provided by Tom Kerppola, University of Michigan School of Medicine, Ann Arbor, MI; Ref. 41). The vectors were transformed into the DH5{alpha} strain of Escherichia coli, sequenced, and purified on two sequential cesium chloride gradients.

Cell Culture and Transient Cotransfections
U2-OS cells were maintained as previously described (8). Transfections were carried out using Lipofectin (Invitrogen, Carlsbad, CA) as described previously (8) with 7.5 µg (Fig. 2Go) or 3.0 µg (Figs. 7Go and 9Go) of the indicated reporter plasmid and 150 ng of the ß-galactosidase vector CMVß-gal (Promega, Madison, WI). Fifty to 250 ng of the c-Fos and c-Jun expression vector (41) and 100 ng of the human ER{alpha} expression vector CMV5hER{alpha} (42) were added as indicated. Cells were maintained in media containing ethanol vehicle or 10 nM E2 for 24 h. ß-Galactosidase activity was determined at room temperature as previously described (43) and used to normalize the amount of CAT activity in each sample. CAT assays were carried out as described (8). PR induction in U2-OS cells was carried out as above except that 1 µg of CMV5hER{alpha} was added to each well of a six-well plate and treated with ethanol or E2 for 24 h.

Gel Mobility Shift Assays
Gel mobility shift assays were carried out essentially as described (7, 8, 44) using the +745 AP-1 containing oligos. 32P-labeled (10,000 cpm) oligos containing the wild-type or mutated +745 AP-1 site were combined with either 20 µg nuclear extract from MCF-7 cells that had been treated with E2 for 0, 2, 24 or 72 h, 5–70 nM purified Fos and Jun protein (kindly provided by Tom Kerppola; Ref. 41) or 100–400 fmol of purified ER{alpha} in binding buffer [15 mM Tris (pH 7.9), 0.2 mM EDTA, 10% glycerol, 50 mM KCl, 1 mM MgCl2, 50 ng of poly(deoxyinosine/deoxycytidine) and 0.4 mM dithiothreitol] for 15 min at room temperature in a final volume of 20 µl. Ovalbumin and KCl were included as needed to maintain constant protein and salt concentrations. BSA was used with purified proteins so that the total protein concentration in each reaction was 20 µg. When MCF-7 nuclear extracts were used, 1 µg of salmon sperm DNA and 2 µg of poly(deoxyinosine/deoxycytidine) were included in each reaction. For antibody supershift experiments, a Fos-, Jun-, or ER{alpha}-specific antibody (sc-52, sc-45, and sc-8005, respectively, from Santa Cruz Biotechnology, Inc., Santa Cruz, CA; except Fig. 3Go where the ER{alpha}-specific antibody, H151, kindly provided by Dean Edwards, University of Colorado Health Science Center, Denver, CO was used) was added to the protein-DNA mixture and incubated for 10 min at room temperature. Low ionic strength gels and buffers were prepared as described (45). Radioactive bands were visualized by autoradiography.

Western Blots
Twenty micrograms of MCF-7 nuclear extract or 15 µg of U2-OS whole cell extract were fractionated on a 10% SDS acrylamide gel and transferred to a nitrocellulose membrane. Primary antibodies to Fos (sc-52, Santa Cruz Biotechnology, Inc.), Jun (sc-45 or sc-822, Santa Cruz Biotechnology, Inc.), and ER{alpha} (sc-8002, Santa Cruz Biotechnology, Inc.), secondary horseradish peroxidase-conjugated antibodies (Zymed, South San Francisco, CA), and Supersignal Luminescent Substrate (Pierce, Rockford, IL) were used for protein detection.

MCF-7 Nuclear Extracts and Purified Fos, Jun, and ER{alpha}
MCF-7 cells were exposed to ethanol or 10 nM E2 for 0, 2, 24, or 72 h, harvested, and processed as previously described (8). The expression and purification of Fos, Jun, and ER{alpha} have been reported (46, 47, 48, 49, 50). The viral stock used for ER{alpha} expression in Sf9 cells was generously provided by J. Kadonaga (University of California, San Diego, CA) and L. Kraus (Cornell University, Ithaca, NY).

ChIP Assays
MCF-7 cells were exposed to ethanol vehicle or 10 nM E2 for 24 or 72 h, and ChIP assays were carried out essentially as described (8). The ER{alpha}-specific antibody RM-9101 (Lab Vision, Fremont, CA), the Fos-specific antibody sc-7202 (Santa Cruz Biotechnology, Inc.), or the Jun-specific antibody sc-1694 (Santa Cruz Biotechnology, Inc.) alone or in combination with sc-45 (Santa Cruz Biotechnology, Inc.) and BD610326 (Biosciences, San Diego, CA) were used for immunoprecipitation of protein-DNA complexes. PCR primers flanking the +745 (5'-TTCTCCTCCCTCTGCCCCTATATTCCCGA-3' and 5'-GGCGACACAGCAGTGGGGAT-3') AP-1 site produced 188-bp DNA fragments. Primers that annealed from -711 to -693 and from -458 to -436 or from -4438 to -4420 and from -4358 to -4336 of the PR gene were used to produce 275- and 103-bp amplified products. These regions of the PR gene do not contain an identifiable ERE or AP-1 site.


   ACKNOWLEDGMENTS
 
We are extremely grateful to Tom Kerppola for providing Fos and Jun expression vectors and purified proteins. We thank Margaret Loven for statistical analysis and Dean Edwards, James Kadonaga, and Lee Kraus for reagents used in these studies.


   FOOTNOTES
 
This research was supported by NIH Grant DK53884 (to A.M.N.). Postdoctoral support for L.P. was provided by NIH Training Grants 2T32 HD 0728-19 and T32 ES07326. J.S. was supported by a Susan G. Komen Breast Cancer Foundation Dissertation Research Award (0201937).

Abbreviations: AP-1, Activator protein-1; CAT, chloramphenicol acetyl transferase; ChIP, chromatin immunoprecipitation; ERE, estrogen response element; E2, 17ß-estradiol; ER, estrogen receptor; PR, progesterone receptor; U2-OS, U2 osteosarcoma.

Received for publication March 26, 2003. Accepted for publication December 10, 2003.


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NURSA Molecule Pages Link:

Nuclear Receptors:   ERα  |  PR
Ligands:   17β-Estradiol



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S. El Marzouk, J. R Schultz-Norton, V. S Likhite, I. X McLeod, J. R Yates, and A. M Nardulli
Rho GDP dissociation inhibitor {alpha} interacts with estrogen receptor {alpha} and influences estrogen responsiveness
J. Mol. Endocrinol., October 1, 2007; 39(4): 249 - 259.
[Abstract] [Full Text] [PDF]

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 Nucleic Acids ResHome page
J. R. Schultz-Norton, V. A. Gabisi, Y. S. Ziegler, I. X. McLeod, J. R. Yates, and A. M. Nardulli
Interaction of estrogen receptor {alpha} with proliferating cell nuclear antigen
Nucleic Acids Res., August 1, 2007; 35(15): 5028 - 5038.
[Abstract] [Full Text] [PDF]

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 Mol. Endocrinol.Home page
J. R. Schultz-Norton, K. A. Walt, Y. S. Ziegler, I. X. McLeod, J. R. Yates, L. T. Raetzman, and A. M. Nardulli
The Deoxyribonucleic Acid Repair Protein Flap Endonuclease-1 Modulates Estrogen-Responsive Gene Expression
Mol. Endocrinol., July 1, 2007; 21(7): 1569 - 1580.
[Abstract] [Full Text] [PDF]

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 Clin. Cancer Res.Home page
V. A. Salvo, S. M. Boue, J. P. Fonseca, S. Elliott, C. Corbitt, B. M. Collins-Burow, T. J. Curiel, S. K. Srivastav, B. Y. Shih, C. Carter-Wientjes, et al.
Antiestrogenic Glyceollins Suppress Human Breast and Ovarian Carcinoma Tumorigenesis
Clin. Cancer Res., December 1, 2006; 12(23): 7159 - 7164.
[Abstract] [Full Text] [PDF]

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 Mol. Endocrinol.Home page
J. R. Schultz-Norton, W. H. McDonald, J. R. Yates, and A. M. Nardulli
Protein Disulfide Isomerase Serves as a Molecular Chaperone to Maintain Estrogen Receptor {alpha} Structure and Function
Mol. Endocrinol., September 1, 2006; 20(9): 1982 - 1995.
[Abstract] [Full Text] [PDF]

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 Biol. Reprod.Home page
C. E. Wood, S. E. Appt, T. B. Clarkson, A. A. Franke, C. J. Lees, D. R. Doerge, and J. M. Cline
Effects of High-Dose Soy Isoflavones and Equol on Reproductive Tissues in Female Cynomolgus Monkeys
Biol Reprod, September 1, 2006; 75(3): 477 - 486.
[Abstract] [Full Text] [PDF]

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 The OncologistHome page
P. D. Ryan and P. E. Goss
Adjuvant Hormonal Therapy in Peri- and Postmenopausal Breast Cancer
Oncologist, July 1, 2006; 11(7): 718 - 731.
[Abstract] [Full Text] [PDF]

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 Genome ResHome page
M. Blanchette, A. R. Bataille, X. Chen, C. Poitras, J. Laganiere, C. Lefebvre, G. Deblois, V. Giguere, V. Ferretti, D. Bergeron, et al.
Genome-wide computational prediction of transcriptional regulatory modules reveals new insights into human gene expression
Genome Res., May 1, 2006; 16(5): 656 - 668.
[Abstract] [Full Text] [PDF]

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 Mol. Endocrinol.Home page
J. Matthews, B. Wihlen, M. Tujague, J. Wan, A. Strom, and J.-A. Gustafsson
Estrogen Receptor (ER) {beta} Modulates ER{alpha}-Mediated Transcriptional Activation by Altering the Recruitment of c-Fos and c-Jun to Estrogen-Responsive Promoters
Mol. Endocrinol., March 1, 2006; 20(3): 534 - 543.
[Abstract] [Full Text] [PDF]

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 Clin. Cancer Res.Home page
H.-J. Kim, X. Cui, S. G. Hilsenbeck, and A. V. Lee
Progesterone Receptor Loss Correlates with Human Epidermal Growth Factor Receptor 2 Overexpression in Estrogen Receptor-Positive Breast Cancer
Clin. Cancer Res., February 1, 2006; 12(3): 1013s - 1018s.
[Abstract] [Full Text] [PDF]

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 Cancer Res.Home page
C. E. Wood, T. C. Register, A. A. Franke, M. S. Anthony, and J. M. Cline
Dietary Soy Isoflavones Inhibit Estrogen Effects in the Postmenopausal Breast
Cancer Res., January 15, 2006; 66(2): 1241 - 1249.
[Abstract] [Full Text] [PDF]

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 JCOHome page
X. Cui, R. Schiff, G. Arpino, C. K. Osborne, and A. V. Lee
Biology of Progesterone Receptor Loss in Breast Cancer and Its Implications for Endocrine Therapy
J. Clin. Oncol., October 20, 2005; 23(30): 7721 - 7735.
[Abstract] [Full Text] [PDF]

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 JNCI J Natl Cancer InstHome page
G. Arpino, H. Weiss, A. V. Lee, R. Schiff, S. De Placido, C. K. Osborne, and R. M. Elledge
Estrogen Receptor-Positive, Progesterone Receptor-Negative Breast Cancer: Association With Growth Factor Receptor Expression and Tamoxifen Resistance
J Natl Cancer Inst, September 7, 2005; 97(17): 1254 - 1261.
[Abstract] [Full Text] [PDF]

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 Mol. Endocrinol.Home page
J. H. Gutzman, S. E. Nikolai, D. E. Rugowski, J. J. Watters, and L. A. Schuler
Prolactin and Estrogen Enhance the Activity of Activating Protein 1 in Breast Cancer Cells: Role of Extracellularly Regulated Kinase 1/2-Mediated Signals to c-fos
Mol. Endocrinol., July 1, 2005; 19(7): 1765 - 1778.
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 JCOHome page
M. C. Gutierrez, S. Detre, S. Johnston, S. K. Mohsin, J. Shou, D. C. Allred, R. Schiff, C. K. Osborne, and M. Dowsett
Molecular Changes in Tamoxifen-Resistant Breast Cancer: Relationship Between Estrogen Receptor, HER-2, and p38 Mitogen-Activated Protein Kinase
J. Clin. Oncol., April 10, 2005; 23(11): 2469 - 2476.
[Abstract] [Full Text] [PDF]

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 Clin. Cancer Res.Home page
C. K. Osborne, J. Shou, S. Massarweh, and R. Schiff
Crosstalk between Estrogen Receptor and Growth Factor Receptor Pathways as a Cause for Endocrine Therapy Resistance in Breast Cancer
Clin. Cancer Res., January 15, 2005; 11(2): 865s - 870s.
[Abstract] [Full Text] [PDF]

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 J. Biol. Chem.Home page
J. R. Schultz, L. N. Petz, and A. M. Nardulli
Cell- and Ligand-specific Regulation of Promoters Containing Activator Protein-1 and Sp1 Sites by Estrogen Receptors {alpha} and {beta}
J. Biol. Chem., January 7, 2005; 280(1): 347 - 354.
[Abstract] [Full Text] [PDF]

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