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Division of Reproductive Biology Research, Department of Obstetrics and Gynecology and Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Northwestern University, Chicago, Illinois 60611
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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decidua, female reproductive tract, implantation, progesterone receptor, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abundant clinical and experimental evidence supports the importance of progesterone in the decidualization process. The effects of progesterone are mediated through interaction with the progesterone receptor (PGR) [4]. The human PGR exists in two isoforms, PGRA and PGRB, which are translated from individual mRNA species of a single gene under the control of distinct promoters [5]. The PGRA gene lacks 164 amino acids from the N-terminus and has been shown to be functionally distinct from the PGRB gene. Both PGRA and PGRB are expressed in the endometrium [6]. A major secretory protein of decidualized endometrial stromal cells, IGFBP1 is activated by progesterone [7–9]. On the IGFBP1 gene promoter, three glucocorticoid receptor (NR3C1, also known as GR)-response elements (GRE) (Fig. 1) have been identified and shown to be the sites responsible for the GR-mediated increase in promoter activity [10]. Both GRE and PGR response elements (PGRE) share the same consensus sequence [11], and Gao et al. [9] demonstrated that the GRE also serve as functional PGRE in endometrial stromal cells.
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Although progesterone is critical during decidualization, it is a weak inducer of the decidual phenotype on its own. The addition of cAMP analogs significantly increase this process (for review, see [2]). Microarray studies show that numerous genes are regulated during decidualization [12– 14]. A member of the FOXO subfamily of the forkhead/ winged-helix family of transcription factors, FOXO1A was one of the earliest induced genes of decidualization [13]. In addition, Christian et al. [15] have shown that cAMP agonists cause increased expression of FOXO1A in the human endometrium and target FOXO1A to the nucleus. These data suggest that one way in which cAMP mediates its effects in the differentiation process is through the action of FOXO1A.
The influence of FOXO1A on the IGFBP1 gene has been studied in liver cells [16–18] and endometrial stromal cells [19]. FOXO1A binds to the insulin-response element (IRE) located in the IGFBP1 proximal promoter region, and it activates promoter activity [16, 18]. Interestingly, contiguous to this sequence is a GRE/PGRE (Fig. 1). FOXO1A has been shown to interact physically with other nuclear-receptor proteins. It interacts with the liganded estrogen receptor 
[20, 21] as well as with the thyroid hormone receptor and retinoic acid receptor [21]. The ability of FOXO1A to associate with a liganded steroid receptor, the close proximity of the IRE and GRE/PGRE, and the ability of both PGR and FOXO1A to activate the IGFBP1 promoter led us to investigate the potential interaction of FOXO1A with PGR. In the present study, we investigated the importance of FOXO1A in IGFBP1 gene transcription, recruitment of PGR to the IGFBP1 gene, and association of PGR with FOXO1A in whole cells. A novel mechanism of IGFBP1 gene regulation, specifically by FOXO1A and PGR, is shown. Parallel studies were done in HEC-1B cells as a comparison of cell-specific responses.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Placental membrane was obtained at the term of pregnancy. Use of these human specimens was approved by the Human Subject Committee of our institution in accordance with U.S. Department of Health regulations. Human endometrial fibroblasts (HuF) were isolated from decidua parietalis dissected from the placental membranes after normal vaginal delivery at term as described previously [22]. Decidualized uterine endometrium maintains a proliferating population of predecidual fibroblastic cells that closely resemble stromal cells [23]. These cells were passaged as needed, up to a maximum of seven passages. Cells were maintained in RPMI-1640 (Invitrogen, Carlsbad, CA) supplemented with sodium pyruvate, penicillin/streptomycin, and 10% fetal bovine serum (FBS; Mediatech, Herndon, VA) that was treated with dextran-coated charcoal (Sigma, St. Louis, MO) according to the manufacturer's protocol to deplete FBS of steroids. At approximately 80% confluence, cells were treated with 36 nM estradiol-17ß, 1 µM medroxyprogesterone acetate (MPA), and 0.1 mM dibutyryl cAMP ((Bu)2cAMP; Sigma) for 10–14 days, with media changed every 2 days. Experiments were repeated using HuF isolated from different placenta. HEC-1B cells, a human endometrial adenocarcinoma cell line, were obtained from American Type Culture Collection (Rockville, MD). Cells were maintained in minimum essential medium (Invitrogen) supplemented with sodium pyruvate, penicillin/streptomycin, and 10% FBS. The T47D cell line (gift from S. Bulun, Northwestern University, Chicago, IL) was maintained in RPMI supplemented with sodium pyruvate, penicillin/streptomycin, and 10% FBS. The T47D cells were serum-starved overnight and incubated with fresh RPMI and 100 nM MPA for 2 h.
Reverse Transcription-Polymerase Chain Reaction
Cells were lysed with TriReagent (Molecular Research Center, Cincinnati, OH). Total RNA was extracted using the protocol provided by the manufacturer. One microgram of total RNA was reverse transcribed, and polymerase chain reaction (PCR) amplification was performed using 2 µl of cDNA for 30 cycles with IGFBP1 primers [3] under the following conditions: 94°C for 1 min, 55°C for 1 min, 72°C for 1.5 min, and a final extension of 72°C for 10 min. The PCR products were electrophoresed in a 1.0% agarose gel, and DNA was visualized by ethidium bromide staining.
Western Blot Analysis
Cells were lysed with RIPA buffer (150 mM NaCl, 1% IGEPAL [Sigma], 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris [pH 8.0]) with protease inhibitors (Sigma) to recuperate whole-cell proteins. Nuclear proteins were isolated from cells using the NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce, Rockford, IL). Protein content was measured using the Micro BCA protein assay kit (Pierce). Freshly isolated proteins were run on a precast, 7.5% acrylamide gel (Bio-Rad, Hercules, CA) and then transferred onto a polyvinylidene fluoride membrane. Membranes were blocked with 5% milk in Tris-buffered saline with 0.1% Tween-20 and then incubated with primary antibody to FOXO1A (FKHR 9462; Cell Signaling, Beverly, MA) or PGR (PGR antibody 1294; gift of D. Edwards, University of Colorado Health Sciences Center, Aurora, CO) [24] followed by incubation with secondary peroxidase-conjugated goat anti-rabbit or anti-mouse immunoglobulin (Ig) G (Bio-Rad). Protein complexes were detected with a chemiluminescent detection kit (Amersham Biosciences, Piscataway, NJ). Membranes were stripped with stripping buffer (Restore; Pierce) and reblotted with a monoclonal antibody to ß-actin (Sigma). For immunoprecipitation studies, 10 µg of a monoclonal PGR antibody (AB-52; Santa Cruz Biotechnology, Santa Cruz, CA) or a purified mouse IgG (Vector Laboratories, Burlingame, CA) were added to 80 µg of fresh nuclear proteins in the presence of protease inhibitors. Protein-antibody complexes were incubated overnight at 4°C on a rotating wheel. Complexes were immunoprecipitated with Protein A Sepharose (CL-4B; Amersham Biosciences), which was washed and prepared in a 50% slurry with PBS. After gentle washing with PBS, Laemmli sample buffer (Bio-Rad) with 2-mercaptoethanol (Bio-Rad) was added to the complexes, boiled for 5 min, and run on a 7.5% acrylamide gel. All Western blots are representative of three independent experiments using cells from three different placentae.
Small Interfering RNA
To silence FOXO1A gene expression, transfection of a small interfering RNA (siRNA) duplex was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol for siRNA. The FOXO siRNA was synthesized by Dharmacon (Lafayette, CO) and corresponded to nucleotides 961–979 of the human FOXO1A coding region (GAGCGTGCCCTACTTCAAG) as described by Potente et al. [25]. The siRNA also targets the sequence for FOXO3. A nonrelated control siRNA that targeted the firefly luciferase protein (catalog no. D-001210-02-05; Dharmacon) was used as a control. Cells were grown in six-well plates until 50% confluence, at which time they were transfected with FOXO siRNA or control siRNA with Lipofectamine 2000. For HEC-1B cells, after 72 h of transfection, cells were lysed with TriReagent for reverse transcription (RT)-PCR analysis or with RIPA buffer for protein analysis. The HuF were transfected for 6 h, after which hormones (36 nM estradiol and 1 µM MPA) and 0.1 mM (Bu)2cAMP were added. Transfection continued for 48 h. Media were changed, and treatment with hormones and (Bu)2cAMP continued for three additional days. Cells were lysed with TriReagent for RT-PCR or with RIPA buffer for protein analysis. Silencing of the FOXO1A gene was verified by Western blot analysis using FOXO1A antibody (Cell Signaling). Both IGFBP1 and GAPDH mRNAs were detected by RT-PCR.
Chromatin Immunoprecipitation
Formaldehyde (Fisher, Fairlawn, NJ) was added directly into cell-culture medium to a final concentration of 1%. Fixation proceeded at room temperature for 10 min and was stopped by addition of glycine to a final concentration of 0.125 M. Dishes were rinsed with cold PBS. Cells were removed by scraping, collected by centrifugation at 2000 rpm for 5 min, and washed in cold PBS. Cells were incubated on ice for 10 min in cell lysis buffer(5 mM PIPES [pH 8.0], 85 mM KCl, 0.5% NP-40, and protease inhibitors). Nuclei were released and collected by microcentrifugation at 4000 rpm for 10 min at 4°C. Pellets were resuspended in nuclear lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris [pH 8.1], and protease inhibitors) and incubated on ice for 10 min. Samples were sonicated on ice to an average length of 300–600 base pairs and then microfuged at 14 000 rpm for 10 min. Samples were diluted fivefold in chromatin immunoprecipitation (ChIP) dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl [pH 8.1], 167 mM NaCl, and protease inhibitors), and the chromatin solution was precleared by incubation with salmon sperm DNA/Protein A agarose (Upstate, Lake Placid, NY) for 1 h at 4°C. Each sample was divided into two aliquots of 500 µl. Each aliquot was mixed with either 5 µg of PGR antibody (AB-52) or purified mouse IgG as a negative control. Incubation occurred overnight at 4°C on a rotating wheel. Forty microliters of protein A agarose were added and incubated for 1 h at 4°C with a standard rotating wheel. Immunoprecipitates were collected by centrifugation at 1000 rpm for 1 min. Pellets were washed twice with low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], and 150 mM NaCl), once with high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], and 500 mM NaCl), four times with LiCl buffer (0.25 M LiCl, 1% IGEPAL-CA630, 1% deoxycholic acid [sodium salt], 1 mM EDTA, and 10 mM Tris [pH 8.1]), and twice with TE buffer (10 mM Tris-HCl and 1 mM EDTA [pH 8.0]). Elution of immune complexes was carried out by addition of 250 µl of elution buffer (1% SDS and 0.1 M NaHCO3) and incubated for 15 min at room temperature on a rotating wheel. Elution was then repeated a second time. After addition of 5 M NaCl, samples were reverse cross-linked by incubation at 65°C for 4 h. Proteins were removed by addition of proteinase K for 1 h at 45°C. The DNA was extracted with phenol:chloroform followed by precipitation with 0.1 volume of 3 mM sodium acetate and 2.5 volumes of ethanol. Pellets were collected by microcentrifugation (14 000 rpm for 15 min), resuspended in 50 µl of water, and analyzed by PCR. The PCR reactions contained 2 µl of DNA, standard PCR reagents, and 50 pmol of each primer: IGFBP1 (left, 5'-GAG ACG CTT TGC AGG AGA [–358 to –341]; right, 5'-TTG CAC CAG GAG GTT AAT GA [–49 to –68]). Negative-control primers, which encompass a sequence distal (–910 to –626) to the putative binding sites, were chosen to monitor specificity of binding (left, 5'-CTCCCTGATCACAGCTCTCC [–910 to –891]; right, 5'-TCTGGAGGGGCAGTTAAGAA [–626 to –645]. After 33 cycles of amplification, PCR products were run on a 1% agarose gel and visualized by ethidium bromide staining.
Reporter Gene Constructs and Expression Vectors
The pHbp1-358.Luc corresponds to the –358 to +75 region relative to the transcription start site of the IGFBP1 gene inserted into the pGL3-basic reporter plasmid (Promega, Madison, WI) as described previously [19]. The pPRE/GRE.E1b.Luc was a gift from M.J. Tsai (Baylor College of Medicine, Houston, TX) and was constructed as described previously [26]. The human FOXO1A expression vector used in these studies is the mutant form in which the three consensus PKB phosphorylation sites (Thr-24, Ser-256, and Ser-319) were mutated to alanines, creating a constitutively active form [16]. This expression vector was given to us by T.G. Unterman (University of Illinois at Chicago, Chicago, IL) [16]. The PGRA and PGRB cDNAs were gifts from P. Chambon (University of Strasbourg, Strasbourg, France). All sequences were verified by dideoxy sequencing.
Cell Transfection and Reporter Gene Studies
Transient transfection of HuF and HEC-1B cells, grown in 12-well plates, was performed using Lipofectamine 2000. Cells were transiently transfected in Dulbecco modified Eagle medium with 1 µg/well of the firefly luciferase reporter plasmid with or without 0.5 µg/well of FOXO1A and/or 0.5 µg/well of PGRA or PGRB expression vectors, along with a ß-galactosidase reporter plasmid (pCMV SPORT; Promega) used as an internal control for normalization. After 4 h, the media were changed to RPMI-1640 with 2% stripped FBS and 100 nM MPA. Cells were incubated for an additional 48 h. Cell extracts were harvested, and luciferase activity was measured with the luciferase reagent kit (Promega). To assess the internal standard activity, ß-galactosidase activity was measured with the ß-Galactosidase Enzyme Assay kit (Promega). Normalized relative luciferase units (RLU) were calculated as firefly luciferase units/ß-galactosidase units. Fold-induction was calculated as normalized RLU of the expression vector divided by normalized RLU of the control (basal activity with no expression vectors). Data are presented as the mean ± SEM of three or more independent experiments, each performed in triplicate.
Statistical Analysis
Multigroup comparisons were done by one-way ANOVA followed by the Newman-Keuls test as post-hoc analysis. Differences were considered to be significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human endometrial fibroblasts were isolated from decidua parietalis dissected from the placental membranes after normal vaginal delivery at term [22]. These fibroblasts are predecidual fibroblastic cells, closely resembling endometrial stromal cells, which exhibit a decidual phenotype in culture when treated with estradiol, progestin, and (Bu)2cAMP for 10–14 days [22, 23]. Treatment of HuF with estradiol, MPA, and (Bu)2cAMP for 10 days (HuF +/+) induced expression of IGFBP1 mRNA, whereas none was detected in the nontreated cells (HuF –/–) (Fig. 2A). The HEC-1B cells, which are an epithelial cell line from an endometrial adenocarcinoma, endogenously expressed IGFBP1 (Fig. 2A). FOXO1A protein was present in the nuclear extracts of both untreated (–/–) and treated (+/+) HuF as well as HEC-1B cells, as demonstrated by Western blot analysis (Fig. 2B). FOXO1A protein levels in HuF +/+ were higher than those of HuF –/– (Fig. 2B). The FOXO1A band for HEC-1B migrated at a higher molecular weight than that of HuF. This product could be a posttranslationally modified form of FOXO1A. Both PGRA and PGRB were expressed in HuF nuclear extracts (Fig. 2C). The level of expression was lower in HuF +/+, which is indicative of down-regulation of receptor expression by the long-term hormone and (Bu)2cAMP treatment. Furthermore, PGRA and PGRB in HuF +/+ were of higher molecular weight, as displayed by their up-shift on the SDS-PAGE gel. This up-shift is similar to that seen in whole-cell extracts of T47D cells treated with MPA (Fig. 2C), which reflects increased phosphorylation at multiple serine residues [27]. HEC-1B cells did not express detectable levels of PGRA or PGRB protein in nuclear protein lysates.
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Silencing FOXO1A Decreases IGFBP1 Gene Expression
Although FOXO1A is up-regulated during decidualization [13, 15, 19] and increases IGFBP1 promoter activity [16, 19], its physiological role in the regulation of IGFBP1 in endometrial cells is unclear. Small interfering RNA specific to FOXO1A [25] was transfected into HuF or HEC-1B cells. Transfection with the FOXO1A siRNA decreased total FOXO1A protein expression in both HEC-1B (Fig. 3A) and HuF (Fig. 3B) compared to that in cells transfected with the control oligo. As a result of FOXO1A silencing, endogenous expression of IGFBP1 mRNA in HEC-1B cells significantly decreased (Fig. 3A). Expression of GAPDH was not affected with FOXO1A siRNA. In HuF, treatment with hormones and (Bu)2cAMP for 5 days induced IGFBP1 mRNA expression, as expected with the control siRNA (Fig. 3B). When FOXO1A was silenced, the expression of IGFBP1 mRNA in response to the hormones significantly decreased. We show here, to our knowledge for the first time, the critical role of FOXO1A in the induction of IGFBP1.
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Recruitment of PGR to IGFBP1 Gene
Ligand-activated PGR mediates many of the progestational effects in endometrium. In the proximal region of the IGFBP1 promoter, three GRE have been identified [10] (Fig. 1). Gao et al. [9] have shown that GRE also are functional PGRE in endometrial cells and that PGR activates the IGFBP1 promoter through at least two of the three PGRE (PGRE1 and PGRE2). To date, and to our knowledge, binding of PGR to the IGFBP1 promoter region in a physiological setting has not been shown. We demonstrate here, by the ChIP technique, recruitment of PGR to the IGFBP1 proximal promoter region (Fig. 4). Human endometrial fibroblasts were treated for 14 days without (–/–) or with (+/+) hormones and (Bu)2cAMP. Cells were fixed, nuclear proteins isolated, DNA sonicated, and complexes immunoprecipitated using a monoclonal PGR antibody or purified mouse IgG. The PCR analysis of the immunoprecipitated complexes showed an amplification of the –358 to –49 region (relative to the transcription start site) of the IGFBP1 promoter for HuF +/+ (Fig. 4). No bands were detected with IgG. Another region of the IGFBP1 promoter, corresponding to –910 to –626, which is distal to the putative binding sites, was amplified to determine specificity of the ChIP assay (Fig. 4). Positive bands were detected in the input samples (total DNA), but no bands were observed for the IgG or PGR antibody immunoprecipitated samples. Thus, ligand-activated PGR was recruited to the –358 to –49 region of the IGFBP1 gene.
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FOXO1A Interacts with PGR
We investigated whether PGR associates with FOXO1A in HuF. Human endometrial fibroblasts were treated without (–/–) or with (+/+) hormones and (Bu)2cAMP for 14 days. Nuclear proteins were isolated and immunoprecipitated using a monoclonal PGR antibody or purified mouse IgG as a control. Complexes were then examined by Western blot analysis using anti-FOXO1A. When HuF nuclear proteins were immunoprecipitated with a PGR antibody, FOXO1A was detected in this complex (Fig. 5). FOXO1A was present in both HuF –/– and HuF +/+. Furthermore, higher levels of FOXO1A were detected in HuF +/+ compared to HuF –/–. No bands were observed with IgG. These results clearly demonstrate that FOXO1A interacts with PGR in HuF. Whether this interaction is direct or indirect (i.e., through the association of other cofactors) remains to be investigated.
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FOXO1A and PGR Regulate Promoter Activity
To determine whether the interaction of FOXO1A and PGR influence transactivation function, FOXO1A and PGRA or PGRB receptor were coexpressed with either a PGRE-linked reporter construct, pPRE/GRE.E1b.Luc [26], or the previously reported IGFBP1 promoter, pHbp1-358.Luc [19]. In all the transfection studies, the human FOXO1A expression vector is the mutant form, in which the three consensus PKB phosphorylation sites (Thr-24, Ser-256, and Ser-319) were mutated to alanines, creating a constitutively active form, thus eliminating possible regulation of translocation by hormones or (Bu)2cAMP. In the absence of MPA, no activation of pPRE/GRE.E1b.Luc occurred in response to PGRA, PGRB, or FOXO1A (results not shown). In the presence of MPA, both PGRA and PGRB increased pPRE/GRE.E1b.Luc activity, with PGRB being the stronger transactivator of this promoter in HuF and HEC-1B cells (Fig. 6). In HuF, when FOXO1A was coexpressed with PGRB, the activity of pPRE/ GRE.E1b.Luc was significantly lower than that of PGRB alone (P < 0.05) (Fig. 6A). No effect of FOXO1A was seen on PGRA transactivation of pPRE/GRE.E1b.Luc. In HEC-1B cells, ligand-activated PGRA and PGRB increased pPRE/GRE.E1b.Luc as expected (Fig. 6B). FOXO1A alone did not increase pPRE/GRE.E1b.Luc activity, but when coexpressed with liganded PGRA or PGRB, the promoter activity increased significantly to levels higher than that of PGR alone, showing an enhancement of PGR-dependent transcription by FOXO1A (P < 0.05).
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The pHbp1-358.Luc reporter (–358 to +75 in pGL3-basic) was cotransfected with PGRA/PGRB and/or FOXO1A. In both HuF and HEC-1B cells, FOXO1A significantly increased activity of pHbp1-358.Luc (Fig. 7). Liganded PGRA alone did not significantly induce promoter activity in either HuF or HEC-1B cells, and liganded PGRB alone was a relatively weak inducer of this promoter. In HEC-1B cells, addition of FOXO1A with PGRA or PGRB increased promoter activity to levels significantly higher than that of FOXO1A alone (P < 0.05) (Fig. 7B). Furthermore, this increase was higher than the sum of the individual responses, suggesting a cooperative increase. In HuF, the combination of FOXO1A and PGRA or PGRB did not increase IGFBP1 promoter activity to levels beyond that of FOXO1A alone (Fig. 7A). The levels were lower or the same as that for FOXO1A, indicating that the combinatorial effect was not additive and suggesting an interaction between the two transcription factors.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Studies have shown that PGR can transactivate the IGFBP1 promoter in endometrial cells [16]. Until now, it has been unclear whether PGR directly binds to chromatin, specifically on the IGFBP1 gene. In the present study, we demonstrate, to our knowledge for the first time, the recruitment of PGR to the proximal promoter region (–358 to –49) of the IGFBP1 gene using the ChIP technique. The PGR is recruited to the IGFBP1 promoter region when cells have been treated with hormones and (Bu)2cAMP, indicating that the liganded PGR is recruited to the promoter. Within this region (–358 to –49), three PGRE have been identified (Fig. 1).
We show in the present study that nuclear FOXO1A and PGR proteins interact with each other in situ both in HuF –/– and HuF +/+. Other studies have shown FOXO1A to interact physically with other transcription factors, albeit in vitro. FOXO1A interacts with liganded estrogen receptor [20], CCAAT/enhancer-binding protein ß [15], thyroid hormone receptor and retinoic acid receptor [21], HOXA5 [31], HOXA10 [19], and peroxisome proliferative activated receptor 
, coactivator 1 [32]. Schuur et al. [20] reported no interaction between FOXO1A and PGR proteins. In their study, interaction was analyzed in vitro using recombinant proteins and the glutathione S-transferase-pulldown method; however, this technique excludes all other molecules within the cells that may be involved in the transcriptional complex. In the present study, the interaction of FOXO1A and PGR was analyzed in vivo. Other factors that may be involved in this complex remain to be identified.
In studies that demonstrate interaction of FOXO1A with other transcription factors, transactivating function is either repressed or stimulated. Here, we show that FOXO1A activates (in HEC-1B) or inhibits (in HuF) PGR transactivation of the PRE-linked reporter, pPRE/GRE.E1b.Luc. Zhao et al. [21] reported a decrease in PRE/GRE-TATA-Luc reporter activity with FOXO1A and PGR in COS-1 cells. This contrasts with the response in HEC-1B cells, in which FOXO1A increased both PGRA and PGRB action. The different responses to FOXO1A and PGR in HEC-1B compared to HuF strongly implicate the involvement of different cofactors in regulating this interaction. HEC-1B cells are an epithelial cell line; thus, the repertoire of cofactors may be quite different from that of a stromal cell line. In addition, because HEC-1B cells do not express endogenous PGR, cofactors of PGR also may be absent or, if present, may not associate with overexpressed PGR, as they do in HuF. In HuF, FOXO1A inhibited PGRB transactivation of pPRE/GRE.E1b.Luc by FOXO1A but not by PGRA. Interestingly, it has been reported that PGRA is the major isoform in decidualized endometrial stromal cells [33, 34]. We can speculate that during decidualization, FOXO1A would not have a significant effect on PGR action (because PGRA is the major isoform expressed) but that PGRA would regulate FOXO1A action. Thus, the inhibiting effect of PGR to FOXO1A activity may be one way in which the decidualization process is slow and controlled, occurring over days of hormone exposure.
The nature of the interaction of FOXO1A with PGR as well as the mechanism by which the two transcription factors influence transactivation are unknown. Moilanen et al. [35] reported the function of one nuclear protein, SNURF, which is able to activate steroid receptor-dependent transcription by forming a functional link between nuclear receptors and other transcription factors (i.e., SP1) [36]. Alternatively, interaction of FOXO1A and PGR could sequester either of these factors and restrict its availability for transactivation function. These studies are currently under investigation.
In summary, we have shown that FOXO1A is critical in the induction of IGFBP1, FOXO1A interacts with PGR, liganded PGR is recruited to the IGFBP1 promoter, and FOXO1A and PGR each regulate the transactivation function of the other. The physiologic consequence of this interaction depends on the cell type. Here, we provide a novel mechanism by which PGR and FOXO1A regulate one decidua-specific gene, IGFBP1, in endometrial cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: J. Julie Kim, Division of Reproductive Biology Research, Department of Obstetrics and Gynecology, Northwestern University, 303 E. Superior Ave., Suite 4-117, Chicago, IL 60611. FAX: 312 503 0095; j-kim4{at}northwestern.edu
Received: 26 April 2005.
First decision: 22 May 2005.
Accepted: 24 June 2005.
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REFERENCES |
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with members of the forkhead transcription factor family. J Biol Chem 2001 276:33554-33560This article has been cited by other articles:
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 |
|
  M. Takano, Z. Lu, T. Goto, L. Fusi, J. Higham, J. Francis, A. Withey, J. Hardt, B. Cloke, A. V. Stavropoulou, et al. Transcriptional Cross Talk between the Forkhead Transcription Factor Forkhead Box O1A and the Progesterone Receptor Coordinates Cell Cycle Regulation and Differentiation in Human Endometrial Stromal Cells Mol. Endocrinol., October 1, 2007; 21(10): 2334 - 2349. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.-H. Chang, T. Chan-Ling, E. L. McFarland, A. Afzal, H. Pan, L. C. Baxter, L. C. Shaw, S. Caballero, N. Sengupta, S. L. Calzi, et al. IGF binding protein-3 regulates hematopoietic stem cell and endothelial precursor cell function during vascular development PNAS, June 19, 2007; 104(25): 10595 - 10600. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. D Rudd, I. Gonzalez-Robayna, I. Hernandez-Gonzalez, N. L Weigel, W. E Bingman III, and J. S Richards Constitutively active FOXO1a and a DNA-binding domain mutant exhibit distinct co-regulatory functions to enhance progesterone receptor A activity J. Mol. Endocrinol., June 1, 2007; 38(6): 673 - 690. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.J. Kim, H.S. Taylor, Z. Lu, O. Ladhani, J.M. Hastings, K.S. Jackson, Y. Wu, S.W. Guo, and A.T. Fazleabas Altered expression of HOXA10 in endometriosis: potential role in decidualization Mol. Hum. Reprod., May 1, 2007; 13(5): 323 - 332. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. S. Jackson, A. Brudney, J. M. Hastings, P. A. Mavrogianis, J. J. Kim, and A. T. Fazleabas The Altered Distribution of the Steroid Hormone Receptors and the Chaperone Immunophilin FKBP52 in a Baboon Model of Endometriosis Is Associated With Progesterone Resistance During the Window of Uterine Receptivity Reproductive Sciences, February 1, 2007; 14(2): 137 - 150. [Abstract] [PDF]
|
|
|
|
|
|
M. C. Jones, L. Fusi, J. H. Higham, H. Abdel-Hafiz, K. B. Horwitz, E. W.-F. Lam, and J. J. Brosens Regulation of the SUMO pathway sensitizes differentiating human endometrial stromal cells to progesterone PNAS, October 31, 2006; 103(44): 16272 - 16277. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Kajihara, M. Jones, L. Fusi, M. Takano, F. Feroze-Zaidi, G. Pirianov, H. Mehmet, O. Ishihara, J. M. Higham, E. W.-F. Lam, et al. Differential Expression of FOXO1 and FOXO3a Confers Resistance to Oxidative Cell Death upon Endometrial Decidualization Mol. Endocrinol., October 1, 2006; 20(10): 2444 - 2455. [Abstract] [Full Text] [PDF]
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 |
|
 O. L. Buzzio, Z. Lu, C. D. Miller, T. G. Unterman, and J. J. Kim FOXO1A Differentially Regulates Genes of Decidualization Endocrinology, August 1, 2006; 147(8): 3870 - 3876. [Abstract] [Full Text] [PDF]
|
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|
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|
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J J Brosens and B Gellersen Death or survival - progesterone-dependent cell fate decisions in the human endometrial stroma. J. Mol. Endocrinol., June 1, 2006; 36(3): 389 - 398. [Abstract] [Full Text] [PDF]
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