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Endocrinology Vol. 148, No. 5 2471-2486
Copyright © 2007 by The Endocrine Society

Distinct Temporal and Spatial Activities of RU486 on Progesterone Receptor Function in Reproductive Organs of Ovariectomized Mice

Sang Jun Han, Sophia Y. Tsai, Ming-Jer Tsai and Bert W. O’Malley

Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Bert W. O’Malley, Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: berto{at}bcm.tmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
RU486 is an incomplete progesterone receptor (PR) antagonist due to its partial agonist activity. To investigate the tissue-specific effects of RU486 on PR function in an ovariectomized mouse model, we used the progesterone receptor activity indicator mouse and evaluated the key determinants of progesterone-dependent gene activity: PR, coregulators, and kinases. As might be expected, acute RU486 treatment (6 h after injection) reduced PR transcriptional activity in the uterus, compared with vehicle or progesterone (P4) treatments. Chronic RU486 treatment (3 d) had a distinctly different effect on PR-mediated transcription, elevating PR activity in both the uterus and mammary gland, whereas chronic P4 treatment reduced PR activity in both tissues. This elevated uterine PR activity was associated with increased modified forms of PR and total protein levels of steroid receptor coactivator (SRC)-1 without affecting SRC-2 or SRC-3 protein levels. In addition to increased levels of coactivators, chronic RU486 treatment activated the ERK-1/2 and c-Jun N-terminal kinase signaling pathways in the uterus in a manner comparable with P4 treatment. In contrast to our observations in the uterus, chronic RU486 treatment increased modified forms of PR and the SRC-3 protein levels (but not SRC-1 and SRC-2 levels) in luminal epithelial cells of the mammary gland. Chronic RU486 also activated the c-Jun N-terminal kinase, but not ERK-1/2, signaling pathways in mammary luminal epithelial cells. This report suggests that in comparison with chronic natural hormone (P4), a mixed antagonist/agonist (RU486) induces a distinct temporal and spatial pattern of cellular genetic regulators that accompany ligand-specific gene expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PROGESTERONE RECEPTOR (PR) plays important roles in regulating homeostasis, female reproductive tissue and mammary gland development, and establishment and maintenance of pregnancy (1, 2, 3, 4, 5). Because tissue-specific modulation of PR activity is a critical issue for women’s health, numerous PR ligands have been synthesized, some of which already have been used for clinical purposes (6, 7). These synthetic PR ligands exhibit a spectrum of activity and range from more pure progesterone (P4) antagonists, such as onapristone (8) and ZK137 316 (9), to mixed agonist/antagonists, which are currently known as selective progesterone receptor modulators (SPRMs), such as 11ß-benzaldoxime-substituted estratrienes (10).

RU486 (mifepristone) is a well-characterized antagonist of PR function. RU486 binds to PR and actively impairs its gene-regulatory activity (11). For this reason, RU486 has been used clinically to prevent PR-dependent cellular processes as a contraceptive and abortive agent (12, 13, 14).

In addition to its PR-antagonistic activity, RU486 also has partial PR-agonist activity (15, 16). For example, RU486 has been clinically used to treat uterine myoma and endometriosis because of its PR-agonistic antiproliferative and antiovulatory effects in a number of species including humans (17, 18). Agonistic activities of RU486 are mediated through the PR-B because such activating effects of RU486 are not detected with the PR-A (16). The partial agonist activity of RU486 is mediated through the N-terminal domain of PR-B. In addition to the N-terminal region of PR-B, other factors contribute to the partial agonistic activity of RU486 in vivo. One such contributing factor is the promoter context of the PR target gene. In response to RU486, PR-B can transactivate a promoter containing a simple progesterone response element up to 20% of that by the full agonist R5020 (16), but under the same conditions, fails to activate the mouse mammary tumor virus promoter, a complex promoter that contains well-defined progesterone response elements (16). Another contributing factor for agonistic activity of RU486 is the cell context. Cell lines derived from different origins each have unique coactivator to corepressor ratios and can thus respond to SPRMs in qualitatively different ways, defining the extent of partial agonistic activity of RU486 in PR-dependent gene regulation (19, 20). Finally, the signaling environment of cells is able to influence RU486-dependent PR activity. For example, the protein kinase A activator 8-bromoadenosine 3',5'-cAMP (8-bromo-cAMP) strongly potentiates the agonist activity of RU486 in cellular milieus in which RU486 is a complete antagonist (21, 22).

These pharmacodynamic properties of RU486 represent an important issue for its usage as a therapeutic agent. However, good animal model systems that could be used to investigate the dynamic properties of RU486 on PR activity are lacking. To investigate the tissue-specific effects of RU486 on PR-dependent gene regulation in vivo, we used the progesterone receptor activity indicator (PRAI) mouse model (23). The PRAI mouse contains a transgenic modified PR bacterial artificial chromosome in which the DNA binding domain of the PR was replaced with the yeast Gal4 DNA binding domain. A humanized green fluorescent protein (hrGFP) reporter controlled by the upstream activating sequences for the Gal4 gene was inserted along with the modified PR gene. The PRAI mouse was shown to replicate faithfully the endogenous pattern of PR and target gene expression in the mouse under various endocrine states (23, 24).

In this study, we used the PRAI mouse model system to investigate new aspects of RU486 on PR activity in reproductive tissues in a mouse model that mimics in part the state of menopause in terms of ovarian steroids. This model allows determination of endogenous PR activity within specific cell types of target organs and temporal comparisons to endogenous regulators of genetic activity. We conclude that chronic treatment with a mixed antagonist/agonist of PR, when compared with natural hormone, results in dramatically different and tissue-specific patterns of intracellular PR activity, coregulator levels and kinase activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals, surgical procedures, and hormone administration
Mice were housed in a pathogen-free animal facility under a standard 12-h light, 12-h dark cycle and fed standard rodent chow and water. All animal experimentation was conducted in accordance with accepted standards of humane animal care. To mimic the menopausal state in the mice, 7-wk-old PRAI female mice were bilaterally ovariectomized. Two weeks after ovariectomy, a daily dose of oil or P4 (12 µmol/kg in sesame oil) or RU486 (12 µmol/kg in sesame oil) was given to PRAI mice (9 wk old) from d 1 to 3. To investigate the acute effect of each hormone on PR activity in uterus and mammary glands, mice were killed 6 h after the first injection and tissues were harvested. To determine the chronic effect of each hormone on PR activity, mice were killed 6 h after the 3-d treatment and the tissues were harvested.

Immunofluorescence analysis
Mice were anesthetized with Avertin and perfused through the heart with 4% paraformaldehyde in PBS. The uterus and mammary gland were removed and kept in the same fixative for 16 h at 4 C. Samples were dehydrated in ethanol, cleared in xylene, and embedded in paraffin. Sections were cut at 7 µm. For immunostaining, sections were dewaxed, rehydrated, and boiled for 10 min in 10 mM citrate buffer (pH 6.0). To reduce nonspecific binding of antibodies, sections were washed in PBS again and preincubated with 5% BSA in PBS for 1 h at room temperature. Antibodies against hrGFP (1:300; Stratagene, La Jolla, CA), PR (1:150 Santa Cruz Biotechnology, Santa Cruz, CA), steroid receptor coactivator (SRC)-1 (1:300; Santa Cruz), SRC-2 (1:500; Bethyl, Montgomery, TX), SRC-3 (1:300; Santa Cruz), phospho-stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) (Thr183/Tyr185; 1:300; Cell Signaling, Beverly, MA), SAPK/JNK (1:300; Cell Signaling), phospho-ERK1 (1:200; Promega, Madison, WI), ERK-1 (1: 200; Promega), phospho-p38 MAPK (Thr180/Tyr182; 1:300; Cell Signaling), and p38 MAPK (1:300; Cell Signaling) were used to detect protein expression in tissues. After probing with primary antibodies, sections were then incubated sequentially with biotinylated horse antirabbit IgG (1:500) and Cy2- or Cy3-conjugated avidin (1:1000; Rockland Inc., Gilbertsville, PA). To determine signal intensity in each compartment, a box was drawn through each compartment in a random orientation. The pixel intensity value of each box (in arbitrary units) was measured and a mean value was obtained. Values shown in all figures are a mean value of intensity (n = 3) ± SD. We used the fluorescence intensity after incubation with normal rabbit serum to normalize the fluorescence intensity of each compartment. The relative fluorescence intensity (RFI) is the ratio of specific antibody fluorescence intensity to normal serum fluorescence intensity for each compartment.

Quantitative real-time PCR
Expression levels of selected genes were validated by real-time RT-PCR TaqMan analysis using the ABI Prism 7700 sequence detector system (PE Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Prevalidated probes and primers for real-time PCR were purchased from Applied Biosystems. cDNA was synthesized using a high-capacity cDNA reverse transcription kit (PE Applied Biosystems). PCR was performed using TaqMan Fast universal PCR master mix reagent and TaqMan gene expression assays (PE Applied Biosystems) according to the manufacturer’s instructions. Standard curves were generated by serial dilution of a preparation of total RNA isolated from each tissue. All real-time PCR was performed by using the three independent RNA sets. mRNA quantities were normalized against 18S RNA using PE Applied Biosystems rRNA control reagents.

Western blot analysis
Uterus and mammary gland tissues were washed with PBS solution and homogenized in a buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2.5 mM EDTA, and 0.125% Nonidet P-40 (vol/vol). Cellular debris was removed by centrifugation at 14,000 rpm for 15 min at 4 C. Protein concentration was determined by Bradford’s method using BSA as the standard. Samples containing 10 µg total proteins were applied to 10% SDS-PAGE. The separated proteins were then transferred onto a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). Membranes were blocked overnight with 5% skim milk (wt/vol) in PBS with 0.1% Tween 20 (vol/vol) (Sigma-Aldrich, St. Louis, MO) and probed with anti-PR and anti-SRC-1 antibody diluted to 1:1000 (Santa Cruz Biotechnology). To determine MAPK signaling, membranes were probed with phospho-SAPK/JNK (Thr183/Tyr185; 1:1000; Cell Signaling), SAPK/JNK (1:1000; Cell Signaling), phospho-ERK1 (1:1000; Promega), ERK-1 (1: 1000; Promega), phospho-p38 MAPK (Thr180/Tyr182; 1:1000; Cell Signaling), and p38 MAPK (1:1000; Cell Signaling). Immunoreactivity was visualized by incubation with a horseradish peroxidase-linked second antibody and treatment with enhanced chemiluminescence reagents. For the normalization, the membrane was stripped, probed with anti-tubulin antibody diluted to 1:1000 (Sigma) at 1:1000 dilution and developed again.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dual RU486 effects on PR activity in the uterus of ovariectomized female mice
To investigate how RU486 impacts PR activity relative to natural hormone in the uterus of ovariectomized mouse, ovariectomized PRAI female mice were treated daily with oil, P4 (12 µmol/kg), or RU486 (12 µmol/kg) for periods of time. The level of PR activity, as determined by reporter hrGFP expression, was assayed by immunohistochemistry using an antibody against hrGFP. As might be expected, PR activity in the uterus was reduced 6 h after the first RU486 injection (Fig. 1Go, A, C, and D), whereas PR activity was increased 6 h after the first P4 injection, compared with oil (Fig. 1BGo). However, in the case of chronic hormone treatment, PR activity was reduced in the uterus after 3 d of P4 treatment, compared with oil (Fig. 1Go, E and F). In contrast to chronic P4 treatment, PR activity was highly elevated in uterus after 3 d of RU486 treatment, compared with oil (Fig. 1Go, G and H). To validate the changing levels of GFP protein in each uterine sample, the level of hrGFP mRNA in each sample was determined by RT-PCR. As shown in Fig. 1IGo, immunofluorescence analysis for hrGFP protein in each sample corresponded to its mRNA level as determined by RT-PCR. Collectively, these data indicate that acute treatment with RU486 antagonized PR activity but chronic RU486 treatment elevated PR-dependent transcription in the uterus of the ovariectomized mouse.


Figure 1
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  		FIG. 1. PR activity in the uterus in response to acute and chronic P4 or RU486 treatment. Two weeks after ovariectomy, daily doses of oil, P4 (12 µmol/kg), or RU486 (12 µmol/kg) were given to wild-type and PRAI mice from d 1 to 3. To determine the effect of acute hormone treatment on PR activity, mice were killed 6 h after the first injection and uterine tissue was collected. hrGFP protein expression in the uterus in response to oil (A), P4 (B), or RU486 (C) treatment for 6 h was examined by immunofluorescence assay. D, The RFI (described in Materials and Methods) value for hrGFP in each compartment of the uterus is reported in the graph. To determine the effect of chronic hormone treatment on PR activity, mice were killed 6 h after the injection on the third day of treatment (3D). hrGFP protein expression in the uterus in response to oil (E), chronic P4 (F), or chronic RU486 (G) treatment was examined by immunofluorescence assay. H, The RFI value for hrGFP in each compartment of uterus is shown in the graph. I, Total uterine RNA was pooled from three mice in each treatment group (3 d treatment; 3D). The cDNA was made from 1 or 4 µg of total RNA from each group. The PCR primer pairs were as follows: 5'-GCT TGG CAT TCC GGT ACT GT-3' and 5'-GGT TGC CGA ACA GGA TGT TG-3' (amplified a 120-bp fragment from hrGFP); 5'-GCA TGG GTC GGG ACA AGA AGA-3' and 5'-CTC CAG CAG GGG GCA CCA CT-3' (amplified a 599-bp fragment from ß-actin). The PCR products were analyzed on a 1.5% agarose gel in Tris-acetate-EDTA buffer. The level of hrGFP mRNA was determined by RT-PCR. RT-PCR for ß-actin mRNA was used for normalization. LE, Luminal epithelium; S, stroma; M, myometrium.

 
PR expression and PR coregulator levels in the uterus are altered by RU486
The above observation raised the question of how RU486 causes a dual effect on PR activity in uterus in our mouse model. To address this question, we investigated the effect of RU486 on the spatial expression of PR in the uterus by immunohistochemistry. Immunofluorescence analyses showed that the PR level in the uterus of ovariectomized mice was not significantly changed 6 h after the first RU486 injection as compared with oil or P4 (Fig. 2Go, A–C). As expected, after 3 d of P4 treatment, PR levels in the luminal epithelium and glandular epithelial (GE) compartments of the uterus were reduced (Fig. 2Go, D–G) (23). In contrast, PR levels in the luminal epithelium and GE compartments of the uterus were not reduced after 3 d of RU486 treatment (Fig. 2Go, H and I). Interestingly, PR levels in both the stroma and myometrium were significantly increased by chronic RU486 treatment, compared with oil or chronic P4 treatment (Fig. 2Go, H and I). To confirm immunostaining results for PR level changes in uterus, Western blot analysis was performed. Consistent with immunostaining results, 1.8- and 3.0-fold increased total PR protein levels were detected in RU486-treated uterus, compared with oil and P4, respectively (Fig. 2KGo). Interestingly, however, PR-A and PR-B were detected as doublets in uterus treated with chronic RU486 and only the modified forms of PR marked with an asterisk were significantly increased by chronic RU486, compared with oil (Fig. 2KGo). To investigate the modified PR in more details, protein extracts made from uteri of PRAI mice treated with RU486 for 3 d were incubated with alkaline phosphatase. After phosphatase treatment, levels of modified PR were significantly reduced (Fig. 2LGo). Thus, modified PR is likely to represent phosphorylated forms of PR. To investigate whether the increased PR protein levels in the uterus (Fig. 2Go, J and K) were associated with increased PR mRNA, real-time RT-PCR was performed with PR-specific primer sets. As shown in Fig. 2MGo, PR mRNA levels in the uterus increased (~2.8-fold) after 3 d of RU486 treatment, compared with oil or P4 treatment. Thus, the increase in uterine PR protein levels during chronic RU486 treatment is associated with elevated expression of its mRNA.


Figure 2
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  		FIG. 2. PR expression in the uterus after P4 or RU486 treatment. PR expression in the uterus in response to oil (A, D, and E), acute P4 (B), acute RU486 (C), chronic P4 (F and G), or chronic RU486 treatment (H and I) was examined by immunofluorescence assay. J, The RFI value for PR in each compartment of the uterus in response to oil, chronic P4, or chronic RU486 treatment is shown in the graph. K, Western blot analysis for PR in the uterus of PRAI mice treated with oil, P4, or RU486 for 3 d. Uterus tissues were lysed, and equal amounts of protein were subjected to SDS-PAGE and Western blot analysis with anti-PR antibody. The asterisk denotes modified PR forms. Total PR levels were quantitated by adding levels of PR-A, PR-B, and modified PRs marked as asterisk in each group. Relative total PR protein levels were quantitated and listed below each panel. L, Western blot analysis for the PR in the RU486-treated uterus before and after alkaline phosphatase treatment. Twenty-five micrograms of protein extracts made from uterus of PARI mice treated with RU486 for 3 d were treated with alkaline phosphatase (Promega) according to its protocol. M, PR mRNA fold induction levels in uterus-treated oil, P4, or RU486 for 3 d. Relative quantification of the mRNA induction levels was accomplished by real-time RT-PCR using PR primer. LE, Luminal epithelium; S, stroma; M, myometrium.

 
Because PR coregulator levels can be dynamically regulated in the uterus and mammary gland after chronic P4 treatment (23, 24), it is possible that PR coregulator levels were altered by RU486 in the uterus. To test this possibility, uterine expression of the primary PR coactivators SRC-1, SRC-2, and SRC-3 as well as PR corepressors nuclear receptor corepressor (NCoR) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) were analyzed by immunofluorescence assay. Acute P4 treatment down-regulates SRC-3 but not SRC-1 and -2 levels in uterus (23). Acute RU486 also down-regulates only SRC-3 level in uterus. Therefore, both acute P4 and RU486 have a similar effect on coactivator levels in uterus (data not shown). In contrast, after 3 d of RU486 treatment, SRC-1 expression was highly elevated in the uterus (Fig. 3Go, A–D). To confirm immunostaining results for change of SRC-1 level in uterus, Western blot analysis was conducted. Consistent with immunostaining data, the total amount of SRC-1 was significantly increased (~2.5-fold) in uterus after 3 d of RU486 treatment as compared with P4 and oil treatment (Fig. 3EGo). Interestingly, in addition to the intact form of SRC-1 (160 kDa), 130- and 90-kDa forms of SRC-1 were detected in uterus treated with P4 or RU486 but not with oil (Fig. 3EGo). To determine whether the elevated SRC-1 protein level in the uterus was associated with increased mRNA expression, real-time RT-PCR was performed using SRC-1-specific primer sets. As shown in Fig. 3FGo, the level of SRC-1 mRNA in the uterus was not significantly changed after 3 d of RU486 treatment when compared with P4 treatment. Therefore, the increased amount of SRC-1 protein in the uterus after chronic RU486 treatment was likely due to a posttranscriptional effect on protein stability. Interestingly, both SRC-2 (Fig. 3Go, G–I) and SRC-3 (Fig. 3Go, J–L) protein levels in the uterus were not changed after 3 d of RU486 treatment as compared with P4 treatment.


Figure 3
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  		FIG. 3. SRC family expression in the uterus in response to P4 or RU486 treatment. SRC-1 expression in the uterus in response to oil (A), chronic P4 (B), or chronic RU486 (C) treatment was examined by immunofluorescence assay. D, RFI values for SRC-1 in each compartment of the uterus are shown in the graph. E, Western blot analysis for SRC-1 in the uterus of PRAI mice treated with oil, P4, or RU486 for 3 d. Uterus tissues were lysed, and equal amounts of protein were subjected to SDS-PAGE and Western blot analysis with anti-SRC-1 antibody. The asterisk denotes isoforms of SRC-1. Total SRC-1 levels were determined by adding levels of all isoforms of SRC-1 in each group. Relative total SRC-1 protein levels were quantitated and listed below each panel. F, SRC-1 mRNA fold induction levels in uterus-treated oil, P4, or RU486 for 3 d. Relative quantification of the mRNA induction levels was accomplished by real-time RT-PCR using SRC-1 primer. SRC-2 expression in the uterus in response to oil (G), P4 (H), or RU486 (I) treatment for 3 d was examined by immunofluorescence assay. SRC-3 expression in the uterus in response to oil (J), P4 (K), or RU486 (L) treatment for 3 d was examined by immunofluorescence assay. LE, Luminal epithelium; S, stroma; M, myometrium.

 
NCoR and SMRT are involved in the repression of PR activity (19), and chronic external hormone treatment can modulate levels of NCoR and SMRT (23). In contrast with that observed for SRC family members, NCoR expression was increased in the uterus 6 h after the first RU486 injection (Fig. 4Go, E and F) as compared with the oil control (Fig. 4Go, A and B) or P4-treated group (Fig. 4Go, C and D). Therefore, increased NCoR protein expression in uterus may contribute to the PR antagonistic activity of mice acutely treated with RU486 (Fig. 4MGo). In contrast to acute RU486 treatment, the level of NCoR protein in the uterus was greatly reduced after chronic RU486 treatment (Fig. 4Go, K and L) as compared with oil (Fig. 4Go, G and H) or P4 (Fig. 4Go, I and J). NCoR mRNA levels in the uterus were not significantly altered after 3 d of RU486 treatment (Fig. 4OGo), indicating that the reduction in uterine NCoR protein expression after chronic RU486 treatment may be due to decreased protein stability (Fig. 4NGo). Collectively, acute RU486 treatment appears to antagonize PR activity in the uterus, possibly by increasing NCoR levels; chronic RU486 treatment agonizes PR activity in uterus accompanied by an increase in the modified form of PRs and the ratio of SRC-1 to NCoR.


Figure 4
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  		FIG. 4. NCoR expression in the uterus in response to P4 or RU486 treatment. NCoR expression in the uterus in response to oil (A, B, G, and H), acute P4 (C and D), acute RU486 (E and F), chronic P4 (I and J), or chronic RU486 treatment (K and L) was examined by immunofluorescence assay. RFI values for NCoR expression in each compartment of the uterus in response to acute hormone treatment (M) or chronic hormone treatment (N) are shown in the graph. O, NCoR mRNA fold induction levels in uterus-treated oil, P4, or RU486 for 3 d. Relative quantification of the mRNA induction levels was accomplished by real-time RT-PCR using NCoR primer. LE, Luminal epithelium; S, stroma; M, myometrium.

 
RU486 stimulates PR activity in the luminal epithelial cells of the mammary gland in ovariectomized female mice
The PR and P4 are centrally involved in mammary gland development (1, 4). To investigate the distinct effects of RU486 on PR activity in the mammary gland, ovariectomized mice were treated with P4 (12 µmol/kg) or RU486 (12 µmol/kg) and compared. PR activity in the luminal epithelial cells (LECs) of mammary gland was assayed by immunohistochemistry using an antibody against hrGFP. The most PR activity was detected in LECs, and little to none was detected in the stromal compartment of the mammary gland. This is in agreement with the PR expression pattern, in that PR is highly expressed in the LECs of the mammary gland but not in every cell (25). PR activity was slightly increased in LECs of mammary gland after 6 h of P4 treatment when compared with the oil control (Fig. 5Go, A, B, and D). However, acute treatment with RU486 significantly increased PR activity in the mammary LECs when compared with oil or P4 treatment (Fig. 5Go, C and D). Therefore, unlike the PR-antagonistic effect of acute RU486 treatment in the uterus, acute RU486 treatment has a PR-agonistic effect in the LECs of the mammary gland.


Figure 5
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  		FIG. 5. PR activity in the LECs of the mammary gland in response to P4 or RU486 treatment. Two weeks after ovariectomy, daily doses of oil or P4 (12 µmol/kg) or RU486 (12 µmol/kg) were given to PRAI mice from d 1 to 3. To determine the effect of acute hormone treatment on PR activity in the mammary gland, mice were killed 6 h after the first hormone injection and the mammary glands were harvested. hrGFP protein expression in the mammary LECs in response to oil (A), P4 (B), or RU486 (C) treatment for 6 h was examined by immunofluorescence assay. D, The RFI value for hrGFP in the LECs of the mammary gland in response to acute each hormone treatment is shown in the graph. To determine the effect of chronic hormone treatment on PR activity, mice were killed 6 h after the injection on the third day of treatment (3D). hrGFP protein expression in the mammary LECs in response to oil (E), P4 (F), or RU486 (G) treatment for 3 d was examined by immunofluorescence assay. H, The RFI value for hrGFP expression in the LECs of the mammary gland treated with each hormone is shown in the graph. LE, Luminal epithelium.

 
In the case of chronic hormone treatment, PR activity in the mammary LECs was clearly reduced after 3 d of P4 treatment (Fig. 5Go, E, F, and H). However, chronic RU486 treatment resulted in markedly elevated PR activity in the LECs of the mammary gland as compared with oil or P4 treatment (Fig. 5Go, G and H). Therefore, unlike in the uterus, both acute and chronic RU486 treatment revealed PR-agonistic activity in mammary tissue.

RU486 alters PR and SRC-3 levels in the LECs of the mammary gland in ovariectomized mice
We previously showed that PR and specific coregulators are dynamically regulated in the LECs of the mammary gland in response to external progesterone stimuli (24). Therefore, we examined the spatial expression of PR and its coregulators in LECs of mammary gland after RU486 treatment. PR expression in the LECs of the mammary gland of our menopausal model mice was not significantly changed 6 h after the first injection of RU486 as compared with oil or P4 (Fig. 6Go, A–C). Therefore, increased PR activity due to acute RU486 treatment is not associated with increased PR expression in the LECs of the mammary gland.


Figure 6
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  		FIG. 6. PR expression in the LECs of the mammary gland in response to P4 or RU486 treatment. PR expression in the LECs of the mammary gland of ovariectomized PRAI mice in response to oil (A and D), acute P4 (B), acute RU486 (C), chronic P4 (E), or chronic RU486 (F) treatment was examined by immunofluorescence assay. G, The RFI values for PR expression in the mammary LECs in mice treated with hormone for 3 d are shown in the graph. H, Western blot analysis for PR in the mammary gland of PRAI mice treated with oil, P4, or RU486 for 3 d. Mammary gland tissues were lysed, and equal amounts of protein were subjected to SDS-PAGE and Western blot analysis with anti-PR antibody. The asterisk denotes modified PR forms. Total PR levels were determined by adding levels of PR-A, PR-B, and modified PRs marked as asterisk in each group. Relative total PR protein levels were quantitated and listed below each panel. I, Western blot analysis for the PR in the RU486-treated uterus before and after alkaline phosphatase treatment. Fifty micrograms of protein extracts made from mammary gland of PARI mice treated with RU486 for 3 d were treated with alkaline phosphatase (Promega) according to its protocol. J, PR mRNA fold induction levels in mammary gland-treated oil, P4, or RU486 for 3 d. Relative quantification of the mRNA induction levels was accomplished by real-time RT-PCR using PR primer. LE, Luminal epithelium.

 
Unlike acute hormone treatment, PR expression in the mammary LECs was clearly reduced after chronic P4 treatment (Fig. 6Go, D and E). The reduction in PR activity in the mammary LECs during chronic P4 treatment is correlated with decreased PR expression. However, PR expression in the mammary LECs was elevated after 3 d of RU486 treatment in comparison with animals treated with P4 (Fig. 6Go, F and G). To confirm immunostaining results for changes in PR levels in mammary glands, Western blot analysis was performed. Consistent with the immunostaining results, 2.3- and 5.7-fold increased total PR protein levels were detected in RU486-treated mammary glands as compared with oil and P4, respectively (Fig. 6HGo). Interestingly, as in the uterus, PR-A and PR-B were detected as a doublet only in mammary glands treated with chronic RU486 and only modified forms of PR marked with an asterisk were significantly increased by chronic RU486, compared with oil and P4 (Fig. 6HGo). To investigate the modified PR in more details, protein extracts made from mammary gland of PRAI mice treated with RU486 for 3 d were incubated with alkaline phosphatase. After phosphatase treatment, modified PR levels were significantly reduced (Fig. 6IGo), indicating that modified PR is likely to represent phosphorylated forms of PR. To investigate whether chronic RU486 treatment impacts transcription of the PR gene, the PR mRNA level in the LECs of the mammary gland was determined (Fig. 6JGo). In contrast with chronic P4, chronic RU486 treatment increased (~3-fold) the PR mRNA level in the LECs of the mammary gland, compared with oil.

We next examined the expression of PR coregulators in the LECs of the mammary gland after RU486 treatment. Expressions of SRC-1, SRC-2, and SRC-3 (Fig. 7Go, A–C) in the mammary LECs were not changed after 6 h of RU486 treatment as compared with oil or P4 treatments (data not shown). Therefore, the increased PR activity after acute RU486 treatment is not associated with increased PR coactivator levels in the LECs of the mammary gland.


Figure 7
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  		FIG. 7. SRC family expression in the LECs of the mammary gland in response to P4 or RU486 treatment. SRC-3 expression in the LECs of the mammary gland of ovariectomized PRAI mice in response to oil (A and D), acute P4 (B), acute RU486 (C), chronic P4 (E), or chronic RU486 treatment (F) was examined by immunofluorescence assay. G, The RFI values for SRC-3 in the LECs of the mammary gland treated with hormone for 3 d are shown in the graph. H, Western blot analysis for SRC-3 in the mammary gland of PRAI mice treated with oil, P4, or RU486 for 3 d. Mammary gland tissues were lysed, and equal amounts of protein were subjected to SDS-PAGE and Western blot analysis with anti-SRC-3 antibody. Relative total SRC-3 protein levels were quantitated and listed below each panel. I, SRC-3 mRNA fold induction levels in mammary gland-treated oil, P4, or RU486 for 3 d. Relative quantification of the mRNA induction levels was accomplished by real-time RT-PCR using SRC-3 primer. SRC-1 expression in the mammary LECs in response to oil (J), P4 (K), or RU486 (L) treatment for 3 d (3D) was examined by immunofluorescence assay. M, The RFI values for SRC-1 are shown in the graph. SRC-2 expression in the mammary LECs in response to oil (N), P4 (O), or RU486 (P) treatment for 3 d was examined by immunofluorescence assay. Q, The RFI values for SRC-2 are shown in the graph. LE, Luminal epithelium.

 
In contrast with acute hormone treatment, the level of SRC-3 protein in the mammary LECs was elevated after 3 d of RU486 treatment in comparison with P4 treatment (Fig. 7Go, D–G). Consistent with immunostaining results, chronic RU486 significantly increased (~2.4-fold) the SRC-3 protein level in mammary gland as compared with oil (Fig. 7HGo). As shown in Fig. 7IGo, the SRC-3 mRNA level was not changed in the LECs of the mammary gland after 3 d of RU486 treatment, compared with oil or P4 treatment. Thus, chronic RU486 treatment appears to increase the level of SRC-3 in the mammary gland by affecting SRC-3 protein stability. However, both SRC-1 (Fig. 7Go, J–M) and SRC-2 (Fig. 7Go, N–Q) protein levels in the LECs of the mammary gland were not changed after 3 d of RU486 treatment. Also, expression of neither NCoR nor SMRT was altered in the mammary LECs in response to acute or chronic hormone treatment (data not shown). Taken together, the elevated PR activity in the LECs of the mammary gland after chronic RU486 treatment was associated selectively with increased modified form of PR and increased SRC-3 protein levels, compared with chronic P4 treatment.

Chronic RU486 treatment activates the ERK1/2 kinase pathway
External signals, such as steroid hormones, can trigger specific kinase signaling pathways to modulate the properties of coregulators, such as cellular localization and stability (26, 27). To determine whether chronic RU486 treatment can activate tissue-specific MAPK signaling pathways that are known to influence coregulator levels/activity, the level of phospho (P)-ERK1/2 in the uterus was examined by immunofluorescence assay. A low level of P-ERK1/2 activity was detected in the uterus of our model mice (Fig. 8AGo) and was not significantly changed after chronic P4 treatment (Fig. 8BGo). In contrast to chronic P4 treatment, the P-ERK1/2 level was highly increased in the uterus after chronic RU486 treatment (Fig. 8CGo). In addition to P-ERK1/2, total ERK1/2 expression pattern was examined by immunofluorescence assay (Fig. 8Go, D–F and O). Collectively, chronic RU486 treatment clearly activated the ERK1/2 signaling pathway in all compartments of the uterus of menopausal model mice when compared with oil or P4 (Fig. 8MGo).


Figure 8
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  		FIG. 8. Activation of the ERK1/2 signaling pathway in the uterus after chronic RU486 treatment. P-ERK1/2 levels in the uterus of ovariectomized PRAI mice in response to oil (A), chronic P4 (B), or chronic RU486 treatment (C) were determined by immunofluorescence assay. The total ERK1/2 expression level in uterus of PRAI mice treated with oil (D), P4 (E), and RU486 (F) for 3 d was determined by immunofluorescence assay. RFI values for p-ERK1/2 (M) and total ERK1/2 (O) level in each compartment of the uterus in response to chronic hormone treatment are shown in graph. The P-ERK1/2 level in the mammary LECs of PRAI mice treated with oil (G), P4 (H), and RU486 (I) for 3 d was determined by immunofluorescence assay. The total ERK1/2 expression level in the LECs of the mammary gland in response to oil (J), P4 (K), and RU486 (L) treatment for 3 d was measured by immunofluorescence assay. RFI values for p-ERK1/2 (N) and total ERK1/2 (P) level in LECs of mammary gland in response to chronic hormone treatment are shown in graph. Q, Western blot analysis confirms increases in ERK1/2 phosphorylation in uterus after chronic RU486 treatment: frozen tissues of untreated or treated PRAI mice were homogenized, lysed, and analyzed by Western blot analysis using a phospho-specific antibody for p-ERK1/2 and total ERK1/2 are shown. Tubulin level in each tissue was determined as a loading control. LE, Luminal epithelium; GE, glandular epithelium; S, stroma; M, myometrium.

 
In contrast to the effects observed in the uterus, the level of P-ERK1/2 in the LECs of the mammary gland was not increased after 3 d of P4 (Fig. 8Go, H and N) or RU486 treatment (Fig. 8Go, I and G). Also, the total ERK1/2 protein level in the LECs of the mammary gland was not changed by chronic P4 or RU486 treatment when compared with oil treatment (Fig. 8Go, J–L and P).

To confirm the activation of the ERK1/2 signaling pathway in uterus, activated ERK1/2 level was determined by Western blot analyses (Fig. 8QGo). Consistent with immunostaining results, only activated ERK1/2 (P-ERK1/2) was detected in uteri treated with RU486 but not in others (Fig. 8QGo).

Chronic RU486 treatment activates the JNK pathway in both the uterus and mammary gland
The P-JNK level in the uterus was not changed during chronic P4 treatment (Fig. 9Go, A, B, and M). In contrast, the P-JNK level in all uterine compartments was highly elevated after chronic RU486 treatment in comparison with the P4 treatment group (Fig. 9Go, C and M). The total JNK level in the GE compartment was elevated by chronic P4 treatment (Fig. 9Go, D, E, and N). However, the total JNK level in other compartments in the uterus was not changed after 3 d of P4 or RU486 treatment when compared with the oil control (Fig. 9Go, D–F and N). Therefore, chronic RU486 treatment activated the JNK signaling pathway in the uterus of our mice, whereas chronic P4 treatment did not (Fig. 9MGo).


Figure 9
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  		FIG. 9. Effect of chronic RU486 treatment on activation of the JNK signaling pathway in the uterus and mammary gland. P-JNK levels in uterus of ovariectomized PRAI mice in response to oil (A), chronic P4 (B), or chronic RU486 treatment (C) were determined by immunofluorescence assay. Total JNK expression in the uterus of PRAI mice treated with oil (D), P4 (E), and RU486 (F) for 3 d (3D) was determined by immunofluorescence assay. RFI values for p-JNK (M) and total JNK (N) level in each compartment of the uterus in response to chronic hormone treatment are shown in graph. The P-JNK level in the mammary LECs of PRAI mice treated with oil (G), P4 (H), and RU486 (I) for 3 d was determined by immunofluorescence assay. The total JNK expression level was measured in mammary gland in response to oil (J), P4 (K), and RU486 (L) treatment for 3 d by immunofluorescence assay. RFI values for p-JNK (O) and total JNK (P) level in LECs of mammary gland in response to chronic hormone treatment are shown in graph. Q, Western blot analysis confirms the induction of JNK phosphorylation after chronic P4 and RU486 treatment: frozen tissues of untreated or treated PRAI mice were homogenized, lysed, and analyzed by Western blot analysis using a phospho-specific antibody for JNK and total JNK are shown. Nonspecific protein detected by antiphospho JNK antibody was marked as asterisk. LE; Luminal epithelium; S, stroma; M, myometrium.

 
The P-JNK level in the LECs of the mammary gland was not increased after 3 d of P4 treatment; however, it did increase after 3 d of RU486 treatment (Fig. 9Go, G–I and O). Total JNK level in LECs of the mammary gland was increased by both chronic P4 and RU486 treatment as compared with oil (Fig. 9Go, J–L and P). Therefore, chronic RU486 treatment activated the JNK signaling pathway in the LECs of the mammary gland of menopausal mice by increasing both P-JNK and total JNK level.

Consistent with immunostaining results, Western blot analysis showed that activated forms of JNK (P-JNK) were elevated in RU486-treated uterus and mammary gland but not in oil- and P4-treated tissues (Fig. 9QGo).

Increased P38 pathway activity in both the uterus and mammary gland in response to chronic P4 treatment
In contrast with ERK1/2 and JNK signaling pathways, the P-P38 level in uterus was highly elevated after 3 d of P4 treatment when compared with oil treatment (Fig. 10Go, A, B, and M). The P-P38 protein level also was increased after 3 d of RU486 treatment in uterus (Fig. 10Go, C and M). However, the total P38 protein level was also elevated in the uterus after 3 d of RU486 treatment when compared with oil or P4 treatment (Fig. 10Go, D–F and N).


Figure 10
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  		FIG. 10. Effect of chronic P4 treatment on activation of the P38 signaling pathway in the uterus and mammary gland. P-P38 levels in the uterus of PRAI mice in response to oil (A), chronic P4 (B), or chronic RU486 treatment (C) were determined by immunofluorescence assay. Total p38 expression level in the uterus of PRAI mice treated with oil (D), P4 (E), and RU486 (F) for 3 d (3D) was determined by immunofluorescence assay. RFI values for P-P38 (M) and total p38 (N) level in each compartment of the uterus in response to chronic hormone treatment are shown in graph. The level of P-P38 in the mammary LECs of PRAI mice treated with oil (G), P4 (H), and RU486 (I) for 3 d was determined by immunofluorescence assay. The total p38 expression level was measured in the mammary LECs in response to oil (J), P4 (K), and RU486 (L) treatment for 3 d by immunofluorescence assay. RFI values for P-P38 (O) and total P38 (P) level in LECs of mammary gland in response to chronic hormone treatment are shown in graph. Q, Western blot analysis confirms the induction of p38 MAPK phosphorylation after chronic P4 and RU486 treatment: frozen tissues of untreated or treated PRAI mice were homogenized, lysed, and analyzed by Western blot analysis using a phospho-specific antibody for P-38 and total P38 are shown. Tubulin level in each tissue was determined as a loading control. LE, Luminal epithelium; S, stroma; M, myometrium.

 
The level of P-p38 in the LECs of the mammary gland also was increased after chronic P4 treatment (Fig. 10Go, G, H, and O). Chronic RU486 treatment slightly elevates the P-p38 level in the mammary LECs (Fig. 10Go, I and O). However, total P38 level in LECs of mammary gland was not altered by chronic P4 and RU486 treatment as compared with oil (Fig. 10Go, J–L and P). Thus, only chronic P4 treatment activated the P38 signaling pathway in the LECs of the mammary gland of ovariectomized mice.

To confirm the activation of P38 signaling in both tissues, activated P38 (P-P38) and total P38 level were determined by Western blot analyses (Fig. 10QGo). Consistent with immunostaining data, activated forms of P38 level (P-P38) were elevated in both P4- and RU486-treated uterus (Fig. 10QGo). In case of mammary gland, activated forms of P38 level (P-P38) were elevated in both P4- and RU486-treated mammary gland. But normalization of P-P38 level with tubulin level showed that P-P38 level in P4-treated mammary gland was higher than in RU486-treated mammary gland (Fig. 10QGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PR plays a central role in the development and function of all female reproductive organs including the uterus and mammary gland. A number of SPRM ligands have been developed with altered biological profiles to meet specific therapeutic needs by modulating PR function in a tissue-specific fashion in vivo. Clearly the regulatory molecules that modulate hormone responsiveness of postmenopausal females needs to be investigated thoroughly to understand the potential mechanism and consequences of therapy with synthetic mixed agonists/antagonists. An important step toward addressing this problem is the development of suitable animal model test systems. To investigate the effect of RU486 on PR activity in a simulated menopausal state (28), our PRAI mouse model system was used to follow endogenous PR activity after ovariectomy (23, 24). This model has been validated for appropriate tissue-specific expression of PR and progesterone-mediated target genes (23, 24).

Tissue-specific effects of RU486 on PR activity
It is well known that selective estrogen receptor modulators (SERMs) regulate estrogen receptor (ER) activity in a tissue-specific fashion in vivo. For example, tamoxifen blocks the action of estrogen through competitive binding to the receptor. However, it is clear that the action of tamoxifen is more complex because this SERM bound receptor also can be transcriptionally active, depending on other factors such as the cellular ratio of expression of coactivators and corepressor (29, 30). We demonstrated that RU486 also can modulate PR activity in a complex manner, with outcomes depending on the specific tissue compartments and the duration of RU486 treatment. Acute RU486 treatment results in PR-antagonistic activity, which is associated with increased NCoR expression in the uterus. In contrast with uterus, acute RU486 treatment led to PR-agonistic activity in the mammary LECs.

Chronic RU486 treatment activates PR activity in both the uterus and mammary gland of menopausal mice
In postmenopausal women treated with estradiol benzoate and mifepristone (RU486) at the dose of 100–200 mg/d, secretory transformation of the endometrium was observed, indicating that RU486 may function as a progesterone agonist (31). Consistent with this finding, we observed that chronic RU486 induction of PR-agonistic activity in the mouse uterus was accompanied by an increase in modified forms of PR and an increase in SRC-1 levels in comparison with chronic P4 treatment. Chronic RU486 treatment also increased PR activity in mammary glands. In contrast to the uterus, the PR-agonistic activity of chronic RU486 treatment in mammary gland was associated selectively with an increase of SRC-3 and modified forms of PR.

RU486 activates a different combination of MAPK signaling pathways in the uterus and mammary gland
The above observation raises the question as to how chronic RU486 treatment specifically increases PR and its tissue-specific coactivator, such as SRC-1 in uterus and SRC-3 in mammary gland. It is known that steroid hormones, such as estrogen and P4, may trigger cell type-specific kinase signaling pathways to modulate cellular processes. For example, estrogen activates multiple signaling pathways, including the MAPK pathway. Activation of the p38 MAPK by estrogen plays key roles in mediating apoptosis, proliferation, and inflammation, all of which take place in the endometrium during cyclical changes under the influence of estrogen (32). In addition to estrogen, RU486 also modulates MAPK signaling to regulate cellular processes. In fact, ERK activation has been shown to be an essential step in the onset of labor in a rat model of preterm labor, and RU486-induced labor is associated with an increase in the active phosphorylated form of ERK2 and increased contractility in vitro (33).

In our model mouse, RU486 also activated MAPK pathways in both uterus and mammary gland. Interestingly, different combinations of MAPKs were activated by RU486, depending on the tissue examined. For example, ERK1/2 and JNK kinase pathways were activated by chronic RU486 treatment in the uterus. However, only the JNK kinase signaling pathway was activated by chronic RU486 in the LECs of the mammary gland. Thus, using different sets of activated MAPKs, RU486 may alter distinct tissue-specific signaling programs that modify its agonist actions in that tissue. Like RU486, P4 also can activate MAPK signaling pathways in both tissues, but the MAPK pathways activated by P4 are different from those activated by RU486. For example, the p38 kinase pathway was activated by chronic P4 treatment but not chronic RU486 treatment in mammary gland. Similarly, RU486 was found to significantly stimulate the activation of p44/p42 MAPKs, whereas P4 markedly inhibited the activation in PR-transfected MDA-MB-231 cells (34). Collectively, by triggering a different set of MAPK pathways, RU486 is thus able to enact a unique biological agonist profile that is qualitatively distinct from P4; this profile is affected by coregulators and is likely to possess characteristics that are either beneficial or deleterious, depending on the tissue and circumstance.

Ligand and kinase signaling pathways can modify PR and alter levels and function of PR coregulators
PR is known to be phosphorylated by various kinases including casein kinase-II, cyclin-dependent kinase-2, and ERK1/2 in response to external hormonal stimuli (35, 36, 37). Chronic RU486 activates specific MAPK combinations in a tissue-specific manner as compared with P4 (Figs. 8–10GoGoGo). Collectively, it is possible that chronic RU486 generates modified forms of PR as a result of increased activated MAPKs. In addition to PR, coactivator level could be modulated by activate MAPK. For example, activated MAPK could involved in coactivator gene expression because elevated SRC-1 RNA level was associated with increased activated ERK1/2 and JNK signaling in RU486-treated uterus (Fig. 3FGo). Alternatively, activating the MAPK also could phosphorylate PR coregulators to modulate their function. SRC-1 has been shown to be phosphorylated by ERK1/2 kinase (38). Also, several different kinases such as JNK, glycogen synthase kinase-3, p38, protein kinase A, ERK, and inhibitor of nuclear factor {kappa} kinase can phosphorylate SRC-3 in response to different stimuli (39). On the basis of the above results, it is our hypothesis that chronic RU486 treatment triggers a specific combination of MAPK pathways, which phosphorylate PR and its coregulators to modulate PR-dependent gene transcription. For example, in T47D cells, RU486 converts to a progesterone agonist in the presence of activators of protein kinase A, such as 8-bromo-cAMP (21, 22, 40). It has been shown that 8-bromo-cAMP potentiation of PR transcriptional activity with RU486 is due to a loss of association with corepressors, such as NCoR and SMRT; 8-bromo-cAMP also induces phosphorylation of two sites in SRC-1 to facilitate progesterone-independent activation of the chicken progesterone receptor (22).

Ligand-dependent down-regulation that leads to rapid and extensive loss of nuclear steroid receptor protein is well recognized. In this process, different phosphorylation pathways may affect the proteasome-mediated degradation of nuclear receptors including ER{alpha} (42) and PR (37). In addition to nuclear receptors, steroid receptor coactivator stability is affected by MAPK signaling. For example, during retinoic acid-dependent activation of the retinoic acid receptor-{alpha} in cultured cells, SRC-3 is phosphorylated by p38 MAPK and upon phosphorylation SRC-3 is subjected to degradation by the proteasome (43). Although not able to be proven in living mouse model, it is possible that chronic RU486 treatment possesses agonist activity primary due to its ability to stabilize coactivator proteins as a result of its ability to influence endogenous kinases and/or the coactivator phosphorylation.

Coactivator stability is also enhanced by the SERM ligands tamoxifen and raloxifene in tissue cultured cells, which may be a similar to that seen here for RU486; SERMs act as agonists in certain circumstances by promoting an increase in the concentration of cellular coactivator proteins (26). Differential expression levels of coactivators and corepressors are major determinants of the tissue-specific agonistic or antagonistic activity of SERM or SPRM (30, 44). For example, in endometrial cells (Ishikawa), tamoxifen acts as an agonist by stimulating recruitment of coactivators to a subset of genes; this does not occur in MCF-7 breast cancer cells because tamoxifen requires the higher level of SRC-1 provided by Ishikawa cells to induce agonist activity (45). Consistent with this interpretation, overexpression of NCoR and/or SMRT can repress the partial agonist activity of tamoxifen-bound ER (30). Similarly, the ability of RU486 to activate transcription was shown to be the direct result of the coactivator to corepressor ratio in a chromatin based cell-free transcription system (20).

Much like our previous findings, which revealed that SERMs can enhance the concentration of SRC-1 and SRC-3 (26), these findings show that in a simulated mouse menopausal model, the SPRM, RU486, can up-regulate the concentration of coactivator proteins in temporally and spatially distinct pattern in PR-target tissues. These findings confirm that the biological activity of mixed antagonist/agonist compounds is complex and not completely predictable. The consequences of coactivator up-regulation clearly can alter endogenous RU486-mediated PR function in a tissue-specific manner (Fig. 11Go). These up-regulated coactivators and the activation of MAPK signaling systems during chronic treatment with RU486 may broadly affect the biology of uterine and breast tissues during administration (Fig. 11Go). Transgenic animal models such as the one we developed and used in this study should shed additional light on the alternations in vivo temporal and spatial tissue patterns of genetic regulations induced by selective receptor modulators and represent an important adjunct to cell-based experimentation.


Figure 11
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  		FIG. 11. Model for RU486 effect on PR activity in uterus and mammary gland of ovariectomized mice. Increased PR activity due to chronic RU486 treatment is associated with elevated active JNK, active ERK-1/2, modified forms of PR, and SRC-1 level in uterus. Decreased NCoR level during chronic RU486 treatment is associated with increased PR activity in uterus. In contrast, increased PR activity due to chronic RU486 treatment is related with elevated active JNK, modified forms of PR, and SRC-3 levels in mammary gland.

 


   Acknowledgments
 
We thank Dr. David Lonard for the review of this manuscript and helpful suggestions.


   Footnotes
 
This work was supported by National Institutes of Health (NIH) Grant P01 DK59820, National Institute of Child Health and Human Development grants, and NIH Grant U19DK062434.

Disclosure Summary: S.J.H., S.Y.T., and M.-J.T. have nothing to declare; B.W.O. has equity interest in Ligand and consults for Wyeth Pharm.

First Published Online February 15, 2007

Abbreviations: 8-Bromo-cAMP, 8-bromoadenosine 3',5'-cAMP; ER, estrogen receptor; GE, glandular epithelial; hrGFP, humanized green fluorescent protein; JNK, c-Jun N-terminal kinase; LEC, luminal epithelial cells; NCoR, nuclear receptor corepressor; P, phospho; P4, progesterone; PR, progesterone receptor; PRAI, progesterone receptor activity indicator; RFI, relative fluorescence intensity; SAPK, stress-activated protein kinase; SERM, selective estrogen receptor modulator; SMRT, silencing mediator of retinoic acid and thyroid hormone receptor; SPRM, selective progesterone receptor modulator; SRC, steroid receptor coactivator.

Received November 27, 2006.

Accepted for publication February 2, 2007.


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 Introduction
 Materials and Methods
 Results
 Discussion
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