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Molecular Endocrinology, doi:10.1210/me.2007-0058
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Molecular Endocrinology 21 (10): 2334-2349
Copyright © 2007 by The Endocrine Society

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

Masashi Takano, Zhenxiao Lu, Tomoko Goto, Luca Fusi, Jenny Higham, Julia Francis, Anna Withey, Jennifer Hardt, Brianna Cloke, Alexandra V. Stavropoulou, Osamu Ishihara, Eric W.-F. Lam, Terry G. Unterman, Jan J. Brosens and J. Julie Kim

Institute of Reproductive and Developmental Biology (M.T., T.G., L.F., A.W., B.C., J.F., J.J.B.) and Cancer Research-UK Labs and Section of Cancer Cell Biology (A.V.S., E.W.-F.L.), Department of Oncology, Imperial College London, Hammersmith Hospital, London W12 OHS, United Kingdom; Department of Obstetrics and Gynaecology (J.Hi.), Imperial College London, St Mary’s Hospital, London W2 1PG, United Kingdom; Department of Obstetrics and Gynecology (O.I.), Saitama Medical School, Moroyama, Saitama 350–0495, Japan; Department of Obstetrics and Gynecology (Z.L., J.Ha., J.J.K), Division of Reproductive Biology Research, Northwestern University, Chicago, Illinois 60611; and Departments of Physiology and Biophysics, and Medicine (T.G.U), University of Illinois at Chicago, College of Medicine and Veterans Affairs Chicago Healthcare System (West Side), Chicago, Illinois 60612

Address all correspondence and requests for reprints to: Julie Kim, Ph.D., Department of Obstetrics and Gynecology, Division of Reproductive Biology Research, Northwestern University, 303 East Superior Street, Lurie 4-117, Chicago, Illinois 60611. E-mail: j-kim4{at}northwestern.edu.; or Jan Brosens, M.D., Ph.D., Institute of Reproductive and Developmental Biology, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, United Kingdom. E-mail: j.brosens{at}imperial.ac.uk.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Differentiation of human endometrial stromal cells (HESCs) into decidual cells is associated with induction of the forkhead transcription factor forkhead box O1A (FOXO1). We performed a genomic screen to identify decidua-specific genes under FOXO1 control. Primary HESCs were transfected with small interfering RNA targeting FOXO1 or with nontargeting control small interfering RNA before treatment with a cAMP analogue and the progestin, medroxyprogesterone acetate for 72 h. Total RNA was processed for whole genome analysis using high-density oligonucleotide arrays. We identified 3405 significantly regulated genes upon decidualization of HESCs, 507 (15.3%) of which were aberrantly expressed upon FOXO1 knockdown. Among the most up-regulated FOXO1-dependent transcriptional targets were WNT signaling-related genes (WNT4, WNT16 ), the insulin receptor (INSR), differentiation markers (PRL, IGFBP1, and LEFTY2), and the cyclin-dependent kinase inhibitor p57Kip2 (CDKN1C). Analysis of FOXO1-dependent down-regulated genes uncovered several factors involved in cell cycle regulation, including CCNB1, CCNB2, MCM5, CDC2 and NEK2. Cell viability assay and cell cycle analysis demonstrated that FOXO1 silencing promotes proliferation of differentiating HESCs. Using a glutathione-S-transferase pull-down assay, we confirmed that FOXO1 interacts with progesterone receptor, irrespectively of the presence of ligand. In agreement, knockdown of PR disrupted the regulation of FOXO1 target genes involved in differentiation (IGFBP1, PRL, and WNT4) and cell cycle regulation (CDKN1, CCNB2 and CDC2) in HESCs treated with either cAMP plus medroxyprogesterone acetate or with cAMP alone. Together, the data demonstrate that FOXO1 engages in transcriptional cross talk with progesterone receptor to coordinate cell cycle regulation and differentiation of HESCs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE POSTOVULATORY RISE in progesterone levels induces extensive remodeling of the endometrium in preparation of a pending pregnancy (1). A cardinal event in this maternal response to pregnancy is the morphological and biochemical differentiation of stromal cells into decidual cells. The extent of the decidual reaction in different species correlates with the depth of placentation, indicating that this process governs trophoblast invasion (2, 3). Furthermore, decidualization of the endometrial stroma ensures tissue homeostasis during the process of endovascular trophoblast invasion, modulates the local immune response at the feto-maternal interface, and confers resistance to oxidative stress (4, 5, 6, 7, 8).

Elevated intracellular cAMP levels and sustained activation of the protein kinase A pathway are a prerequisite for human endometrial stromal cells (HESCs) to decidualize in response to progesterone (9). Multiple mechanisms have been described by which cAMP signaling sensitizes HESCs to progesterone-dependent differentiation. First, progesterone exerts its action on decidualizing cells predominantly by activating the progesterone receptor A (PR-A) isoform, a member of the superfamily of ligand-activated transcription factors (10, 11). Activation of the protein kinase A pathway by cAMP has been shown to disrupt the interaction of PR with the corepressors NCoR and SMRT and facilitates the recruitment of coactivators such as SRC-1 (12, 13). Second, cAMP signaling attenuates ligand-dependent SUMOylation of PR-A, which in turn greatly enhances the transcriptional activity of this receptor isoform (14). Furthermore, cAMP induces the expression or activation of several transcription factors, including p53, forkhead box O1A (FOXO1), STAT5, and C/EBPß, capable of interacting, directly or indirectly, with PR-A (9, 15, 16, 17, 18). Thus, by interacting with cAMP-induced transcription factors, the activated PR-A may acquire control of a myriad of genes, many devoid of consensus progesterone response elements (PREs) in their promoter regions.

FOXO1, a member of the FOXO sub-family of Forkhead/winged helix family of transcription factors, is one of the earliest induced transcription factors in HESCs in response to cAMP signaling (15, 19). FOXO proteins, FOXO1, FOXO3a, and FOXO4, are evolutionarily conserved transcriptional activators of genes involved in cell cycle inhibition (e.g. CDKN1B, also termed p27Kip1) (20), induction of apoptosis such as BCL2L11 (BIM), FASLG (Fas ligand or CD95L) and TNFSF10 (TRAIL) (21, 22), defense against oxidative stress (e.g. SOD2 and CAT), and DNA repair (e.g. GADD45A) (23, 24, 25). FOXO proteins are downstream of the phosphatidylinositol-3-kinase (PI3K) signaling pathway and targeted phosphorylation of these transcription factors by activated Akt (also termed PKB) triggers their export from the nucleus and binding to 14–3-3 chaperone proteins in the cytosol (26, 27). In addition to Akt, other kinases, such as SGK1 (serum- and glucocorticoid-inducible kinase 1), CK1 (casein kinase 1), and DYRK1A (dual-specificity tyrosine-phosphorylated and regulated 1A), have also been implicated in FOXO phosphorylation and nuclear export (28, 29, 30).

Progesterone signaling enhances cAMP-dependent induction of FOXO1 in differentiating HESCs (1, 31). However, progestins like MPA (medoxyprogesterone acetate) also elicit a partial translocation of this transcription factor to the cytoplasm of decidualizing cells in a PI3K-dependent manner. Nevertheless, there is strong evidence to suggest that the residual nuclear pool of transcriptionally competent FOXO1 is important for the expression of the decidual phenotype. For instance, FOXO1 interaction with PR and HOXA10 or C/EBPß has been shown to induce the expression of IGFBP1 (IGF binding protein 1) and PRL (prolactin), respectively, by binding to defined composite response elements in the proximal promoter regions of these decidua marker genes (15, 16, 32). Furthermore, FOXO1 has also been implicated in the regulation of genes involved in extracellular matrix remodeling, such as DCN (decorin), TIMP3 (tissue inhibitor of metalloproteinase 3), and LEFTY2 (left-right determination factor 2, also known as endometrial bleeding-associated factor (TGFß-4) (33, 34).

In this study, we used genome-wide microarray analysis to interrogate the expression of genes perturbed upon silencing of FOXO1 expression in decidualizing HESCs. We identified 507 FOXO1-dependent decidua-specific genes, many of which are involved in cellular differentiation and cell cycle regulation. Moreover, we demonstrate that silencing of PR expression impairs the expression of selected FOXO1 target genes in HESCs treated with cAMP plus MPA or cAMP alone. Together, the data demonstrate that the transcriptional cross talk between FOXO1 and PR governs diverse facets of decidualizing human endometrium.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of FOXO1 Target Genes in Decidualizing HESCs
We combined a gene knockdown approach with high-density oligonucleotide microarray analysis to identify FOXO1-dependent genes in decidualizing HESCs. To this end, three separate primary HESC cultures were first transfected with either a pool of nontargeting (NT) oligonucleotides or with a small interfering RNA (siRNA) pool targeting FOXO1. Subsequently, cells remained untreated or differentiated with cAMP plus MPA for 72 h. Parallel cultures were harvested for mRNA and protein analysis. As shown in Fig. 1AGo, FOXO1 was induced at mRNA and protein level upon treatment with cAMP plus MPA in HESCs transfected with NT siRNA. However, transfection of FOXO1 siRNA not only abolished this induction but the level of FOXO1 expression was below that observed in undifferentiated cells. Notably, a modest reduction in basal FOXO1 mRNA levels, ranging between 15 and 30%, was also noted in undifferentiated cells transfected with NT siRNA when compared with mock transfected cells (Fig. 1AGo).


Figure 1
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  		Fig. 1. FOXO1 Knockdown in Decidualizing HESCs and Microarray Analysis

A, FOXO1 knockdown in differentiating human endometrium. The upper panel shows RTQ-PCR analysis of FOXO1 transcript levels in mock-transfected HESCs or cells transfected with NT or FOXO1 siRNA. Subsequently, cells remained untreated or differentiated cAMP and MPA for 72 h as indicated. The relative expression of FOXO1 transcripts was normalized to that of L19 mRNA, and the results show the mean (±SD) of three separate cultures, each measured in triplicate. The lower panel shows Western blot analysis of FOXO1 protein in whole cell lysates obtained from parallel cultures. ß-actin served as a loading control. B, Venn diagram showing the number of genes regulated at least 1.5-fold in a statistically significant manner and the overlap between groups. The diagram summarizes the following pair-wise comparisons: 1) mock transfected undifferentiated cells vs. decidualizing HESCs transfected with NT siRNA; 2) mock vs. NT siRNA transfected undifferentiated HESCs; and 3) decidualizing HESCs transfected with NT siRNA vs. FOXO1 siRNA.

 
Total RNA was then processed for analysis on whole genome, high-density human genome U133 Plus 2.0 Arrays (Affymetrix, Inc., Santa Clara, CA), containing 42,203 genes and 12,397 expressed sequence tags, and the data interrogated using parametric statistical testing. Differentially expressed genes were defined by a lower boundary of a 95% confidence interval of fold change greater than 1.5. Figure 1BGo represents the Venn diagram of regulated genes identified in the following pair-wise comparisons: 1) mock transfected undifferentiated cells vs. decidualizing HESCs transfected with NT siRNA; 2) mock vs. NT siRNA transfected undifferentiated HESCs; and 3) decidualizing HESCs transfected with NT siRNA vs. FOXO1 siRNA.

As shown in Fig. 1BGo, 281 genes were found to be regulated upon transfection of NT siRNA when compared with mock transfected HESCs, and these were excluded from further analysis. Treatment of HESCs with cAMP plus MPA for 72 h was associated with altered expression of 3307 genes, 507 (15.3%) of which were regulated in a FOXO1-dependent manner. Interestingly, the expression of a comparable number of genes (n = 503) not regulated in response to cAMP and progesterone signaling was also perturbed in cells transfected with FOXO1 siRNA. Out of the 3307 genes associated with HESC differentiation, 1247 (38%) were up-regulated and 2060 (62%) down-regulated. Supplemental Table 1 (published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org) lists the 50 most up- and down-regulated genes, many of which have been identified in previous studies (35, 36). Among the decidua-specific genes, 328 (10%) were up-regulated and 179 (5.4%) down-regulated in a FOXO1-dependent manner. Tables 1Go and 2Go list the top 50 up-regulated and down-regulated FOXO1-dependent genes, respectively, and the entire gene list is presented in supplemental Table 2. Interestingly, 12 of the 50 most highly induced genes in HESCs treated with cAMP plus MPA (ranging from 8- to 157-fold) were FOXO1-dependent. Even more strikingly, 60% of genes repressed more than 8-fold upon decidualization were perturbed upon FOXO1 silencing. Cluster analysis of the 507 genes is depicted in Fig. 2Go. It is apparent that FOXO1 is indispensable for the induction of many highly expressed genes in decidualizing HESCs, while it simultaneously represses an even larger number of genes. Furthermore, the apparent discrepancy between the relatively modest induction of FOXO1 and its profound effect on decidual gene expression supports the notion that additional factors modify the transcriptional competence of FOXO1 in decidualizing cells.


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  		Table 1. Top 50 Genes Induced in Decidualizing Cells in a FOXO1-Dependent Manner

 

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  		Table 2. Top 50 Genes Repressed in Decidualizing Cells in a FOXO1-Dependent Manner

 

Figure 2
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  		Fig. 2. Overview of Expression Patterns of 507 Genes Regulated upon cAMP plus MPA Treatment in a FOXO1-Dependent Manner

Each row is a standardized gene expression profile. The rows were sorted by one-dimensional MDS. The red represents high expression, the green represents low expression, and the yellow represents close to median expression.

 
Validation of FOXO1-Dependent Gene Expression Involved in Decidualization
We used real-time quantitative PCR (RTQ-PCR) to validate putative novel FOXO1-dependent genes that are regulated upon decidualization. Independent primary HESC cultures were first mock transfected or transfected with FOXO1 siRNA, differentiated with a cAMP analog and MPA, and total mRNA harvested after 72 h. We quantified the expression of genes that were highly induced (e.g. WNT4), modestly induced (e.g. BAMBI, ATP8A2, RBBP6), or repressed (e.g. NEK2, CDCA1, CCNB2, and CDC2) upon decidualization. As shown in Fig. 3Go, silencing of FOXO1 perturbed the level of expression of all eight transcripts, in agreement with the microarray results.


Figure 3
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  		Fig. 3. Validation of Novel FOXO1 Target Genes Involved in Decidualization

Primary cultures were transfected with or without FOXO1 siRNA and treated with cAMP plus MPA for 72 h as indicated. Levels of WNT4 (A), BAMBI (B), ATP8A2 (C), RBBP6 (D), NEK (E), CDCA1 (F), CCNB2 (G), and CDC2 (H) transcripts were measured by RTQ-PCR. The data were normalized either to L19 or 36B4 transcript levels, as indicated, and expressed as fold change compared with the expression level of untreated cells. Results are the mean (±SD) of three separate cultures, each measured in triplicate. *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

 
However, some previously identified FOXO1 target genes, such as SOD2 and BCL2L11 (BIM) (23), did not reach the statistical threshold of the array analysis, largely because of variation in signal intensities between samples. Hence, RTQ-PCR was also used to validate the expression of various known FOXO1 target genes, including IGFBP1, PRL, SOD2, BCL2L11, CNR1 and LEFTY2, as well as CDKN1C (p57Kip2), a gene previously reported to be regulated by HOXA10 in uterine stromal cells (37, 38). As shown in Fig. 4Go, the induction of all these genes in response to cAMP and MPA was unequivocally abolished or attenuated upon silencing of FOXO1 expression by siRNA.


Figure 4
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  		Fig. 4. RTQ-PCR Validation of Known FOXO1 Target Genes in Differentiating HESCs

Primary cultures were transfected with or without FOXO1 siRNA and treated with cAMP plus MPA for 72 h as indicated. Levels of IGFBP1, PRL, SOD2, BCL2L11, CDKN1C, LEFTY, and CNR1 transcripts were measured by RTQ-PCR. The data were normalized either to L19 or 36B4 transcript levels, as indicated, and expressed as fold change compared with the expression level of untreated cells. Results are the mean (±SD) of triplicate measurements of a representative experiment. **, P < 0.01; and ***, P < 0.001.

 
The magnitude of gene regulation was broadly similar between the microarray and RTQ-PCR analyses, although a notable exception was CNR1, which encodes for the cannabinoid receptor 1. Based on the array, CNR1 was by far the most induced gene in decidualizing cells (505-fold, supplemental Table 1), which was much higher than determined by RTQ-PCR (22-fold; Fig. 4Go). Furthermore, RTQ-PCR demonstrated that FOXO1 knockdown elicited a 4-fold increase in CNR1 transcript levels, although this was not apparent from the array data. The reason for this discrepancy is not clear. Thus, RTQ-PCR validation uncovered some false-negative but no false-positive results, suggesting that the array analysis may not have yielded the full spectrum of FOXO1-regulated genes in decidualizing HESCs.

FOXO1 Inhibits Cell Cycle Progression in Decidualizing HESCs
Decidual FOXO1-dependent genes were further annotated in silico using gene ontology and Ingenuity Pathways Analysis (IPA; Ingenuity Systems, Redwood City, CA) (Fig. 5Go). This analysis revealed that a preponderance of FOXO1-regulated genes was involved in cell cycle progression (e.g. CCNB1, CCNB2, CDC2, BIRC5, NEK2, CDKN1C; Table 3Go), DNA replication (e.g. MCM5 and CENPF), and mitosis (e.g. PRC1, NUSAP1, SPBC25, and ASPM). This expression profile strongly suggested a role for FOXO1 in modulating decidual cell proliferation. To test this, primary HESC cultures were first transfected with either NT or FOXO1 siRNA and re-plated after 48 h in 96-well plates at a density of 5 x 104 cells/ml. The cultures were treated with cAMP and MPA, and the proportion of viable cells measured by MTS assay over the following 2 d. Figure 6AGo demonstrates the relative change in cell number after 24 and 48 h of treatment. Differentiated HESCs proliferate slowly as demonstrated by the little or no increase in cell number in mock transfected cells treated with cAMP plus MPA. However, FOXO1 knockdown significantly increased the number of cells over time. Consistent with this, flow cytometry analysis demonstrated no change in the fraction of apoptotic cells (<2N) but an increase (mean ± SD: 29 ± 5%; n = 3) in the proportion of differentiating cells in the S and G2/M phases upon FOXO1 knockdown (Fig. 6BGo).


Figure 5
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  		Fig. 5. Ingenuity Pathway Analysis of Functional Categories for the 507 FOXO1-Dependent Decidua-Specific Genes

Functional categories (molecular and cellular) that contain a significant number of differentially expressed genes at a threshold of > -(log) 1.25 are shown. The numbers on the x-axis refer to the following processes or functions: 1) Cell cycle; 2) Cellular Assembly and Organization; 3) DNA replication, Recombination and Repair; 4) Cellular Movement; 5) Cellular Compromise; 6) Cell Death; 7) Gene Expression; 8) Cell Morphology; 9) Cellular Growth and Proliferation; 10) Carbohydrate Metabolism; 11) Small Molecule Biochemistry; 12) Energy Production; 13) Nucleic Acid Metabolism; 14) Cellular Development; 15) Amino Acid Metabolism; 16) Lipid Metabolism; 17) Post-Translational Modification; 18) Cell Signaling; 19) RNA Post-Transcriptional Modification; 20) Molecular Transport; 21) Protein Synthesis; 22) Cell-to-Cell Signaling and Interaction; 23) Cellular Function and Maintenance; 24) Cellular Response to Therapeutics; 25) Drug Metabolism; 26) Protein Trafficking; and 27) Vitamin and Mineral Metabolism.

 

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  		Table 3. FOXO1-Dependent Genes Involved in Cell Cycle Regulation

 

Figure 6
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  		Fig. 6. FOXO1 Knockdown Enhances Cell Cycle Progression in Differentiating HESCs

A, HESC cells transfected with FOXO1 siRNA (dotted line) or control siRNA (solid line) were plated in 96-well plates, treated after 1 d with cAMP plus MPA. Cell viability was measured 24 and 48 h later (d 2 and 3, respectively). The results show the relative fold change in viable cells compared with untreated cells 24 h after plating. The data presented are the mean (±SD) of triple measurements. ***, P < 0.001. B, HESCs transfected with NT or FOXO1 siRNA and treated with cAMP plus MPA were analyzed by flow cytometry. The percentage of cell in each phase of the cell cycle (<2N, G0/G1, S, G2/M) is indicated. One representative result of three independent experiments is shown.

 
Regulation of FOXO1-Dependent Genes in Decidualizing HESCs Requires PR
We have previously reported that FOXO1 co-immunoprecipitates with PR in nuclear extracts of differentiating HESCs, irrespectively of the presence or absence of progesterone (16). As shown in Fig. 7AGo, in vitro protein binding studies confirmed direct interaction between glutathione-S-transferase (GST) tagged full-length FOXO1 (GST-FOXO1) and wild-type (wt) PR-A. GST-FOXO1 also interacted with a PR-A ligand binding domain mutant (PR-ALBM), in which the ligand binding domain was truncated at position 645. Notably, the interaction between GST-FOXO1 and wt PR-A but not PR-ALBM was enhanced in the presence of MPA.


Figure 7
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  		Fig. 7. FOXO1 Engages in Transcriptional Cross Talk with PR

A, In vitro translated 35S-labeled PR-A and PR-ALBM were incubated with GST and GST-FOXO1 immobilized on gluthatione-Sepharose, as indicated. Ligand activation of 35S-labeled receptor was performed by preincubation with 1 µM MPA for 2 h at 4 C. Specifically bound proteins were resolved on 10% SDS-PAGE and visualized by autoradiography. B, PR knockdown perturbs the expression of FOXO1 target genes involved in decidualization. Primary HESCs transfected with control or PR siRNA were treated with cAMP, MPA, or a combination for 48 h. The cultures were then harvested and mRNA expression analyzed by RTQ-PCR. Expression of IGFBP1, PRL, WNT4, CDKN1C, CCNB2, and CDC2 transcripts was normalized to 36B4 mRNA, and the data represent fold regulation relatively to expression levels in untreated HESCs transfected with control siRNA. Data are a representative of three independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

 
These observations raised the possibility that PR serves as a co-regulator for FOXO1 in HESCs, even in the absence of progesterone signaling. To test this, we transfected primary cultures with siRNA targeting PR or NT siRNA. Transfection of cells with PR siRNA attenuated the expression of PR. Also, levels of nuclear FOXO1 in response to cAMP signaling did not change upon PR knockdown (data not shown). Next we examined the consequences of PR knockdown on the expression of six FOXO1 decidual target genes, two of which are repressed (CCNB2 and CDC2) and the remainder induced (IGFBP1, PRL, WNT4, and CDKN1). Treatment with cAMP alone was sufficient to induce or repress these decidua-specific FOXO1 target genes, although the level of regulation was greatly enhanced upon co-treatment with MPA (Fig. 7BGo). As expected, the fold induction or repression of all genes in response to cAMP and MPA signaling was significantly reduced by 30–60% in differentiating cells transfected with PR siRNA. More strikingly, expression was also perturbed upon PR knockdown in cells stimulated with cAMP alone. Thus the data indicate that PR, even in the absence of ligand, serves as a co-regulator of FOXO1-dependent genes in differentiating HESCs.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Based on previous microarray studies, decidualization has been described as a process of sequential reprogramming of functionally related families of genes involved in extracellular matrix organization, cell adhesion, cytoskeletal organization, signal transduction, metabolism, differentiation, and apoptosis (19, 36). Our observations concur with this view. In fact, manual mining of the Endometrium Database Resource (http://endometrium.bcm.tmc.edu/edr/edr_home.do) revealed that a substantial proportion (25%) of highly regulated decidua-specific genes had already been identified in other studies, despite differences in experimental design and the use of more limited arrays. The gene expression profile also highlights the importance of the decidual process in modulating immune and oxidative stress responses at the feto-maternal interface. For example, HESC differentiation is associated with strong induction of HSD11B1 expression (105-fold), which encodes for 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1). This enzyme operates predominantly as reductase that converts inactive cortisone into active cortisol. In contrast, 11ß-HSD2, which inactivates cortisol, localizes to placental syncytiotrophoblast (35). The distinct expression pattern of these two isoenzymes suggests the presence of a cortisol gradient at the feto-maternal interface, which could constitute a major mechanism that protects the fetal allograft against a potential maternal immune response. GPX3, encoding for gluthatione peroxidase 3 (GPx3), also ranks among the most highly regulated genes upon decidualization of HESCs (20-fold). GPx3 is a secreted enzyme with potent extracellular antioxidant activity capable of reducing hydrogen peroxide and a broad range of fatty acid- and phospholipid-hydroperoxides (39, 40). Hence, the decidual process may contribute to a local microenvironment that protects the invading trophoblast against the high levels of reactive oxygen species produced during the process of spiral artery remodeling (41, 42, 43).

Decidualization can also be viewed as a process of epitheloid transformation of endometrial fibroblasts into secretory cells. In fact, a myriad of genes (e.g. SOD2, GADD45A, MAOB, SSP1, CLU and FOXO1) are either constitutively or transiently expressed in the epithelial cell compartment after ovulation before being induced in differentiating stromal cells during the mid- to late-secretory phase of the cycle (23, 31, 44, 45, 46). Expression of several of these genes (e.g. SOD2, MAOB and CLU) was perturbed upon FOXO1 knockdown in differentiating HESCs, suggesting a pivotal role for this transcription factor in the acquisition of an epitheloid phenotype. Moreover, FOXO1 regulates the expression of major secretory products of decidual cells (e.g. IGFBP1, PRL, LEFTY2, and WNT4) and overexpression of a constitutively active FOXO1 mutant has been shown to induce a decidual morphology in untreated HESCs (33).

A striking observation is that FOXO1 co-ordinates the expression of gene networks associated with cellular differentiation while simultaneously repressing gene clusters involved in cell cycle progression. Proliferation requires successful progression through the cell cycle, a tightly regulated process divided into four phases: an S (DNA-synthesis) phase and M (mitosis) phase, which are separated by two intervals, G1 (gap 1-between M and S) and G2 (gap 2-between S and M) phases (47, 48). In other cell systems, FOXO transcription factors have been shown to induce G1 cell cycle arrest by activating genes that encode for negative cell cycle regulators, including CDKN1A (p21Cip1/WAF1), CDKN1B (p27Kip1), and RBL2 (RB2/P130), and by repressing the expression of genes important for G1 progression, such as CCND1 (cyclin D1) and CCND2 (cyclin D2) (49, 50, 51, 52). In differentiating HESCs, however, FOXO1 regulates the induction of CDKN1C (p57Kip2), a cyclin-dependent inhibitor involved in G1 arrest. More strikingly, FOXO1 also represses several genes important for either DNA replication/S phase (e.g. MCM5), G2/M transition (e.g. CCNB1, CCNB2, CDC2, BIRC5, and BRIP1) or mitosis (e.g. PRC1, NUSAP1, CENPF, SPBC25, and ASPM). This profile concurs with our observation that FOXO1 knockdown induces proliferation of differentiating HESCs by increasing the proportion of cells in the S/G2/M phases of the cell cycle. Together, the data point toward a major role for FOXO1 in ensuring endometrial homeostasis and, in agreement, impaired FOXO1 expression has been associated with proliferative endometrial disorders, such as endometriosis and endometrial cancer (53, 54).

FOXO transcription factors integrate various signal transduction pathways. They not only function downstream of the PI3K pathway but also bind directly to Smad3 and Smad4 in response to TGF-ß signaling (52, 55). Furthermore, ß-catenin, which accumulates in the nucleus upon activation of the canonical WNT pathway, also binds directly to FOXO proteins and enhances FOXO transcriptional activity in mammalian cells (56). Our microarray analysis now reveals that FOXO1 in turn regulates the expression of several genes capable of modulating the activities of these upstream regulatory pathways. For instance, the FOXO1-dependent induction of insulin receptor mRNA (INSR, 10-fold) suggests that PI3K signaling may be amplified in decidualizing cells. Even more pronounced is the induction of WNT4 expression (157-fold). WNT4 is a critical mediator of progesterone-dependent mammary gland development (57), although its role in decidualization of human endometrium remains to be defined. FOXO1 also induces the expression of BAMBI (bone morphogenic protein and activin membrane-bound inhibitor), which in other cell systems has been reported to be a downstream target in the ß-catenin and TGF-ß pathways (58, 59). BAMBI is a transmembrane glycoprotein related to TGF-ß family type I receptors. However, it lacks an intracellular kinase domain and serves as a potent inhibitor of bone morphogenic protein, activin, and TGF-ß signaling (60). Thus, the profiling of FOXO1 target genes in decidualizing HESCs strongly points toward the existence of complex feedback mechanisms that determine the preference and fine-tune the activity of upstream regulatory pathways.

Although FOXO1 is induced in HESCs in response to cAMP, progesterone signaling is a major determinant of its transcriptional output. On the one hand, progestins like MPA elicit a partial translocation of FOXO1 to the cytoplasm (31). Previously we demonstrated that withdrawal of MPA from differentiated HESC cultures results in rapid nuclear accumulation of FOXO1, activation of the proapoptotic B-cell CCL/lymphoma 2 family member BIM, and cell death. Silencing of FOXO1 expression abolishes entirely the induction of cell death in differentiated HESCs upon progesterone withdrawal, thereby providing a possible mechanistic link between the decidual process and shedding of the superficial endometrium in response to falling progesterone levels at the end of the menstrual cycle (31). On the other hand, immunoprecipitation experiments demonstrated that FOXO1 is complexed to PR in the nuclear fraction of HESCs, independently of the presence or absence of hormone (16). We substantiated this observation by demonstrating that direct interaction between FOXO1 with PR-A does not require a functional ligand binding domain in the receptor. However, MPA enhanced the interaction between these transcription factors and, in agreement, amplified the induction as well as the repression of FOXO-1-dependent genes. Interestingly, knockdown of PR not only perturbs the expression of FOXO1-dependent genes in cells treated with cAMP and MPA but also in cultures treated with cAMP alone. Together, the data indicate that FOXO1 engages in transcriptional cross talk with PR upon cAMP signaling. Although this cross talk is not strictly dependent upon ligand, interaction and transcriptional co-operation with FOXO1 is more efficient with hormone-bound PR. The finding that the unliganded PR has a transcriptional role in HESCs is akin to observations made in breast cancer cells, where induction of PR in the absence of hormone, especially PR-A, suffices to alter the expression of entire gene networks (61).

In summary, this study demonstrates that cross talk between PR and FOXO1 underpins the decidualization process in human endometrium. While others have provided gene array studies regarding the global picture of gene expression that occurs during decidualization, this study now demonstrates how FOXO1 and PR function cooperatively and play a major role in the regulation of multiple pathways that are critical for promoting the process of decidualization. Our data also revealed that FOXO1 plays an essential role in coordinating different aspects of this process, including cell proliferation, differentiation, immune modulation, and defenses against environmental or oxidative stress, as well as engaging in a complex feedback with several upstream regulatory pathways in decidualizing HESCs. For the first time, we show a role for the unliganded PR in the transcriptional regulation of decidua-specific genes. Together, these data provide important novel insights into the mechanisms that underpin the decidual process in humans. Aberrant expression or activity of such an important intermediate of endometrial progesterone responses may not only result in a hostile uterine environment for pregnancy but also contribute to the development of uterine pathologies such as endometriosis, endometrial cancer, and leiomyomas.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Experiments were performed at Imperial College London as well as at Northwestern University in Chicago. Although the results were consistent, minor variations in materials and methods between our institutions have been highlighted.

Cell Culture
Human endometrial tissue was obtained from hysterectomies or biopsies from pre-menopausal women with no clinically documented abnormalities of the endometrium. This study was approved by the Human Subject Committee at Northwestern University, in accordance with U.S. Department of Health regulations, as well as by the Ethics Committees of Hammersmith and St. Mary’s Hospitals, London, United Kingdom. HESCs were isolated as previously described (16, 62, 63). Cells were grown until 80% confluence and subsequently remained untreated or treated with 1 µM MPA, with 0.5 mM of a cAMP analog (db-cAMP, Sigma, St. Louis, MO; or 8-br-cAMP, Sigma-Aldrich Co., Ltd., Dorset, UK), alone or in combination. This hormonal treatment is referred to as cAMP and MPA.

Small Interfering RNA and Transient Transfection
Transfections were carried out using either the ProFection mammalian transfection kit (Promega Corp., Southhampton, UK; Imperial College London) or Lipofectamine 2000 (Invitrogen, Carlsbad, CA; Northwestern University). In knockdown experiments, HESCs were transiently transfected with 50 nM of the following siRNA reagents purchased from Dharmacon: FOXO1 siGENOME SMARTpool, siCONTROL Non-Targeting siRNA Pool, or PR siGENOME SMARTpool (23, 31). After siRNA transfection, the medium was changed to 2% charcoal-stripped FBS and cells treated with MPA, cAMP, or a combination for 48 or 72 h as described previously (15, 62).

Western Blot Analysis
Whole-cell extracts or nuclear and cytoplasmic protein fractions were immunoblotted as described (31, 33). The following primary antibodies were used: rabbit polyclonal anti-FOXO1 (Cell Signaling Technology, Danvers, MA), PR (Novocastra Laboratories Ltd, Newcastle upon Tyne, UK) and ß-actin (Abcam, Cambridge, UK). The primary antibodies were used at 1:1000 except for the antibody to ß-actin (diluted 1:100000). Protein complexes were detected with a chemiluminescent detection kit (Amersham Biosciences, Piscataway, NJ). When appropriate, membranes were stripped with stripping buffer (100 mM 2-ß-mercaptoethanol; 2% sodium dodecyl sulfate weight/vol; 6.25 mM Tris-HCl, pH 6.7) and reprobed.

Microarray Analysis and Statistical Analysis
Three separate primary HESC cultures derived from three different patients were used for microarray analysis. Each culture consisted of four experimental conditions; hence, a total of 12 microarray chips were used. The experimental conditions were: 1) mock transfected (no siRNA oligos); 2) transfected with a pool of NT oligonucleotides; 3) transfected with NT oligos and treated with cAMP and MPA; and 4) transfected with a siRNA pool targeting FOXO1 with subsequent treatment of cAMP and MPA. All RNA samples were processed at the Microarray Core Facility in the Center for Genetic Medicine at Northwestern University (Chicago, IL). The quality of total RNA was evaluated using Bioanalyzer 2100 (Agilent Technologies, Inc., Santa Clara, CA). 1.5 µg of each RNA sample, with 260/280 and 28S/18S ratio of greater than 1.8, was used to make double-stranded cDNA and labeled cRNA following the One-Cycle Target Labeling Assay from Affymetrix. The size distribution and fragmentation quality of the biotin-labeled cRNA were checked by the Bioanalyzer 2100. The fragmented labeled cRNA was hybridized to the Human U133-Plus 2.0 Array (Affymetrix, Inc.) for 18 h. The chips were then scanned, the data extracted using the Affymetrix GeneChip Operating Software (Affymetrix, Inc.), and analyzed further by use of GeneSpring software (Agilent Technologies, Inc.). The normalized data were analyzed by pair-wise comparisons to create a list of differentially expressed genes. Gene expression levels were quantified using the RMA algorithm with the quantile normalization built in. Data analysis was conducted using the Bioconductor/R package (64, 65). To find statistically consistent genes of differential expression, we used a linear model with empirical Bayesian correction, and changes in transcript levels of at least 1.5-fold were validated by Student’s t test (P < 0.05). To interpret the biological significance of differentially expressed genes, a gene ontology analysis was conducted using DAVID/EASE (66) and Ingenuity Pathways Analysis (IPA, Ingenuity Systems).

RTQ-PCR
Total RNA samples were DNase treated to remove any contaminating DNA. Briefly, the volume of RNA for 1 µg of cDNA was added to 10 x Dnase 1 reaction buffer (1 µl), Dnase 1 Amp grade (1 µl), and made up to 10 µl with DEPC treated water. The solution was incubated at room temperature for 15 min, the reaction was quenched by addition of 25 mM EDTA (1 µl) and heated for another 10 min at 65 C. Total RNA was reverse transcribed in a total volume of 20 µl. RTQ-PCR analysis was performed at both institutions, using either SYBR green fluorescence or Taqman. Each real-time PCR reaction consisted of 1 µl RT product, 10 µl SYBR Green or Taqman PCR Master Mix (PE Applied Biosystems, Foster City, CA), and 500 nM forward and reverse primer pairs: L19-sense (5'- GCG GAA GGG TAC AGC CAA T-3') and L19-antisense (5'-GCA GCC GGC GCA AA-3'); 36B4-sense (5'-GAC ACC CTC CAG GAA GCG A-3') and 36B4-antisense (5'-GTG TTC GAC AAT GGC AGC AT-3'); FOXO1-sense (5'-TGG ACA TGC TCA GCA GAC ATC-3') and FOXO1-antisense (5'-TTG GGT CAG GCG GTT CA-3'); IGFBP1-sense (5'-CGA AGA CTC TCC ATG TCA CCA-3') and IGFBP1-antisense (5'-TGT CTC CTG TGC CTT GGC TAA AC-3'); PRL-sense (5'-AAG CTG TAG AGA TTG AGG AGC AAA C-3') and PRL-antisense (5'-TCA GGA TGA ACC TGG CTG ACT A-3'); WNT4-sense (5'-GGA ACA AGC AGA TAC CAG GTC AA-3') and WNT4-antisense (5'-TAT CGA ACC TCT AGC TGT CCA TGT AA-3'); BAMBI-sense (5'-GAA AAT AAG AGG CTG CAG GAT CA-3') and BAMBI-antisense (5'-GGA ATG GTG TCC GTG AAA GC-3'); CDKN1C-sense (5'-GCC TCT GAT CTC CGA TTT CTT C-3') and CDKN1C-antisense (5'-GAC ATC GCC CGA CGA CTT-3'); SOD2-sense (5'-AAT TGC TGC TTG TCC AAA TCA G-3') and SOD2-antisense (5'-TCC CCA GCA GTG GAA TAA GG-3'); BCL2L11-sense (5'-CAC AAA CCC CAA GTC CTC CTT –3') and BCL2L11-antisense (5'-TTC AGC CTG CCT CAT GGA A-3'); CDKN1C-sense (5'-GCC TCT GAT CTC CGA TTT CTT C-3') and CDKN1C-antisense (5'-GAC ATC GCC CGA CGA CTT-3'); CNR1-sense (5'-AAG ACG GTG TTT GCA TTC TG-3') and CNR1-antisense (5'-GTC GCA GGT CCT TAC TCC TC-3'). Primer sets of Taqman gene expression assay kit (Applied Biosystems, Foster City, CA) were used for the mRNA detection of ATP8A2, RBBP6, CCNB2, CDC2, NEK2, and CDCA1. All reactions were carried out on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems) for 40 cycles (95 C for 15 sec, 60 C for 1 min) after 10-min incubation at 95 C. Data analysis was done by two different methods, depending on the institution where the PCR was done. One method used the relative standard curves method (Imperial College London). L19, a nonregulated ribosomal housekeeping gene, served as an internal control and was used to normalize for differences in input RNA (23). The other method was to calculate the data as fold change in expression of each gene using the {Delta}{Delta}Ct method (Northwestern University), with the ribosomal protein 36B4 mRNA as an internal control (67). All values are presented as mean ± SD, and the data were analyzed using two-tailed t test with values of P < 0.05 considered significant.

Proliferation and Cell Cycle Analysis
Primary undifferentiated HESCs were transfected with NT or FOXO1 siRNA in six-well plates. Cells were then re-plated after 48 h in 96-well plates at a density of 5 x 104 cells/ml and treated with or without cAMP plus MPA for 72 h. Two hundred microliters of CellTiter 96 Aqueous one solution cell proliferation assay (Promega Corp., Madison, WI) were added to each well and incubated at 37 C in 5% CO2 atmosphere. After 2 h the absorbance was recorded on a 450 Bio-Rad microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA) at 490 nm. All values are presented as mean ± SD, and the data were analyzed using two-tailed t test with values of P < 0.05 considered significant. Flow cytometry analysis of ethanol-fixed, propidium iodide-stained cells was used to determined the proportion of cells in different phases of the cell cycle as described previously (68). All experiments were performed on a minimum of three independent cultures.

GST Pull-Down Assays
GST pull-down assays were performed as described (15, 63). 35S-labeled proteins were prepared by the in vitro transcription-translation method, using the TNT T7 Coupled Reticulocyte Lysate System following the supplier’s protocol (Promega). The presence of 35S-methionine (>1000Ci/mmol, Amersham Biosciences, Piscataway, NJ) in the incubation mixture was used to produce labeled wt PR-A and PR-ALBM from pSG/hPR-A and pSG/hPR-ALBM expression vectors, respectively.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Simon Lin and Dr. Pan Du at the Bioinformatics Center at Northwestern University for analyzing the microarray data. We would also like to thank Dr. Nadereh Jafari and her team at the Genomics Core Facility at Northwestern University for performing the microarray technique with our RNA samples.


   FOOTNOTES
 
This work was supported by Grant HD044715 from the National Institutes of Health and a grant from the Friends of Prentice (to J.J.K) and by grants from the Great Britain Sasakawa Foundation and the Institute of Obstetrics and Gynaecology Trust (to J.J.B.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 3, 2007

Abbreviations: BAMBI, Bone morphogenic protein and activin membrane-bound inhibitor; FOXO1, forkhead box O1A; GST, glutathione-S-transferase; HESC, human endometrial stromal cells; IGFBP1, IGF binding protein 1; MPA, medroxyprogesterone acetate; NT, nontargeting; PI3K, phosphatidylinositol-3-kinase; PR, progesterone receptor; PRL, prolactin; RTQ-PCR, real-time quantitative PCR; siRNA, small interfering RNA; wt, wild type.

Received for publication January 30, 2007. Accepted for publication June 29, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Brosens JJ, Gellersen B 2006 Death or survival–progesterone-dependent cell fate decisions in the human endometrial stroma. J Mol Endocrinol 36:389–398[Abstract/Free Full Text]
  2. Brosens JJ, Pijnenborg R, Brosens IA 2002 The myometrial junctional zone spiral arteries in normal and abnormal pregnancies: a review of the literature. Am J Obstet Gynecol 187:1416–1423[CrossRef][Medline]
  3. Ramsey EM, Houston ML, Harris JW 1976 Interactions of the trophoblast and maternal tissues in three closely related primate species. Am J Obstet Gynecol 124:647–652[Medline]
  4. Kliman HJ 2000 Uteroplacental blood flow. The story of decidualization, menstruation, and trophoblast invasion. Am J Pathol 157:1759–1768[Free Full Text]
  5. Kudo Y, Hara T, Katsuki T, Toyofuku A, Katsura Y, Takikawa O, Fujii T, Ohama K 2004 Mechanisms regulating the expression of indoleamine 2,3-dioxygenase during decidualization of human endometrium. Hum Reprod 19:1222–1230[Abstract/Free Full Text]
  6. Red-Horse K, Drake PM, Fisher SJ 2004 Human pregnancy: the role of chemokine networks at the fetalmaternal interface. Expert Rev Mol Med 2004:1–14[Medline]
  7. Sherwin JR, Sharkey AM, Cameo P, Mavrogianis PM, Catalano RD, Edassery S, Fazleabas AT 2007 Identification of novel genes regulated by chorionic gonadotropin in baboon endometrium during the window of implantation. Endocrinology 148:618–626[Abstract/Free Full Text]
  8. Sugino N, Shimamura K, Takiguchi S, Tamura H, Ono M, Nakata M, Nakamura Y, Ogino K, Uda T, Kato H 1996 Changes in activity of superoxide dismutase in the human endometrium throughout the menstrual cycle and in early pregnancy. Hum Reprod 11:1073–1078[Abstract/Free Full Text]
  9. Gellersen B, Brosens J 2003 Cyclic AMP and progesterone receptor cross-talk in human endometrium: a decidualizing affair. J Endocrinol 178:357–372[Abstract]
  10. Lydon JP, DeMayo FJ, Funk CR, Mani SK, Hughes AR, Montgomery Jr CA, Shyamala G, Conneely OM, O’Malley BW 1995 Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities. Genes Dev 9:2266–2278[Abstract/Free Full Text]
  11. Tseng L, Zhu HH 1997 Regulation of progesterone receptor messenger ribonucleic acid by progestin in human endometrial stromal cells. Biol Reprod 57:1360–1366[Abstract]
  12. Rowan BG, Garrison N, Weigel NL, O’Malley BW 2000 8-Bromo-cyclic AMP induces phosphorylation of two sites in SRC-1 that facilitate ligand-independent activation of the chicken progesterone receptor and are critical for functional cooperation between SRC-1 and CREB binding protein. Mol Cell Biol 20:8720–8730[Abstract/Free Full Text]
  13. Wagner BL, Norris JD, Knotts TA, Weigel NL, McDonnell DP 1998 The nuclear corepressors NCoR and SMRT are key regulators of both ligand- and 8-bromo-cyclic AMP-dependent transcriptional activity of the human progesterone receptor. Mol Cell Biol 18:1369–1378[Abstract/Free Full Text]
  14. Jones MC, Fusi L, Higham JH, Abdel-Hafiz H, Horwitz KB, Lam EW, Brosens JJ 2006 Regulation of the SUMO pathway sensitizes differentiating human endometrial stromal cells to progesterone. Proc Natl Acad Sci USA 103:16272–16277[Abstract/Free Full Text]
  15. Christian M, Zhang X, Schneider-Merck T, Unterman TG, Gellersen B, White JO, Brosens JJ 2002 Cyclic AMP-induced forkhead transcription factor, FKHR, cooperates with CCAAT/enhancer-binding protein ß in differentiating human endometrial stromal cells. J Biol Chem 277:20825–20832[Abstract/Free Full Text]
  16. Kim JJ, Buzzio OL, Li S, Lu Z 2005 Role of FOXO1A in the regulation of insulin-like growth factor-binding protein-1 in human endometrial cells: interaction with progesterone receptor. Biol Reprod 73:833–839[Abstract/Free Full Text]
  17. Mak IY, Brosens JJ, Christian M, Hills FA, Chamley L, Regan L, White JO 2002 Regulated expression of signal transducer and activator of transcription, Stat5, and its enhancement of PRL expression in human endometrial stromal cells in vitro. J Clin Endocrinol Metab 87:2581–2588[Abstract/Free Full Text]
  18. Pohnke Y, Schneider-Merck T, Fahnenstich J, Kempf R, Christian M, Milde-Langosch K, Brosens JJ, Gellersen B 2004 Wild-type p53 protein is up-regulated upon cyclic adenosine monophosphate-induced differentiation of human endometrial stromal cells. J Clin Endocrinol Metab 89:5233–5244[Abstract/Free Full Text]
  19. Brar AK, Handwerger S, Kessler CA, Aronow BJ 2001 Gene induction and categorical reprogramming during in vitro human endometrial fibroblast decidualization. Physiol Genomics 7:135–148[Abstract/Free Full Text]
  20. Dijkers PF, Medema RH, Pals C, Banerji L, Thomas NS, Lam EW, Burgering BM, Raaijmakers JA, Lammers JW, Koenderman L, Coffer PJ 2000 Forkhead transcription factor FKHR-L1 modulates cytokine-dependent transcriptional regulation of p27(KIP1). Mol Cell Biol 20:9138–9148[Abstract/Free Full Text]
  21. Modur V, Nagarajan R, Evers BM, Milbrandt J 2002 FOXO proteins regulate tumor necrosis factor-related apoptosis inducing ligand expression. Implications for PTEN mutation in prostate cancer. J Biol Chem 277:47928–47937[Abstract/Free Full Text]
  22. Sunters A, Fernandez de Mattos S, Stahl M, Brosens JJ, Zoumpoulidou G, Saunders CA, Coffer PJ, Medema RH, Coombes RC, Lam EW 2003 FoxO3a transcriptional regulation of Bim controls apoptosis in paclitaxel-treated breast cancer cell lines. J Biol Chem 278:49795–49805[Abstract/Free Full Text]
  23. Kajihara T, Jones M, Fusi L, Takano M, Feroze-Zaidi F, Pirianov G, Mehmet H, Ishihara O, Higham JM, Lam EW, Brosens JJ 2006 Differential expression of FOXO1 and FOXO3a confers resistance to oxidative cell death upon endometrial decidualization. Mol Endocrinol 20:2444–2455[Abstract/Free Full Text]
  24. Kops GJ, Dansen TB, Polderman PE, Saarloos I, Wirtz KW, Coffer PJ, Huang TT, Bos JL, Medema RH, Burgering BM 2002 Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature 419:316–321[CrossRef][Medline]
  25. Nemoto S, Fergusson MM, Finkel T 2004 Nutrient availability regulates SIRT1 through a forkhead-dependent pathway. Science 306:2105–2108[Abstract/Free Full Text]
  26. Biggs 3rd WH, Meisenhelder J, Hunter T, Cavenee WK, Arden KC 1999 Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1. Proc Natl Acad Sci USA 96:7421–7426[Abstract/Free Full Text]
  27. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, Blenis J, Greenberg ME 1999 Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96:857–868[CrossRef][Medline]
  28. Brunet A, Park J, Tran H, Hu LS, Hemmings BA, Greenberg ME 2001 Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a). Mol Cell Biol 21:952–965[Abstract/Free Full Text]
  29. Rena G, Woods YL, Prescott AR, Peggie M, Unterman TG, Williams MR, Cohen P 2002 Two novel phosphorylation sites on FKHR that are critical for its nuclear exclusion. EMBO J 21:2263–2271[CrossRef][Medline]
  30. Woods YL, Rena G, Morrice N, Barthel A, Becker W, Guo S, Unterman TG, Cohen P 2001 The kinase DYRK1A phosphorylates the transcription factor FKHR at Ser329 in vitro, a novel in vivo phosphorylation site. Biochem J 355:597–607[CrossRef][Medline]
  31. Labied S, Kajihara T, Madureira PA, Fusi L, Jones MC, Higham JM, Varshochi R, Francis JM, Zoumpoulidou G, Essafi A, Fernandez de Mattos S, Lam EW, Brosens JJ 2006 Progestins regulate the expression and activity of the forkhead transcription factor FOXO1 in differentiating human endometrium. Mol Endocrinol 20:35–44[Abstract/Free Full Text]
  32. Kim JJ, Taylor HS, Akbas GE, Foucher I, Trembleau A, Jaffe RC, Fazleabas AT, Unterman TG 2003 Regulation of insulin-like growth factor binding protein-1 promoter activity by FKHR and HOXA10 in primate endometrial cells. Biol Reprod 68:24–30[Abstract/Free Full Text]
  33. Buzzio OL, Lu Z, Miller CD, Unterman TG, Kim JJ 2006 FOXO1A differentially regulates genes of decidualization. Endocrinology 147:3870–3876[Abstract/Free Full Text]
  34. Grinius L, Kessler C, Schroeder J, Handwerger S 2006 Forkhead transcription factor FOXO1A is critical for induction of human decidualization. J Endocrinol 189:179–187[Abstract/Free Full Text]
  35. Alfaidy N, Li W, MacIntosh T, Yang K, Challis J 2003 Late gestation increase in 11ß-hydroxysteroid dehydrogenase 1 expression in human fetal membranes: a novel intrauterine source of cortisol. J Clin Endocrinol Metab 88:5033–5038[Abstract/Free Full Text]
  36. Tierney EP, Tulac S, Huang ST, Giudice LC 2003 Activation of the protein kinase A pathway in human endometrial stromal cells reveals sequential categorical gene regulation. Physiol Genomics 16:47–66[Abstract/Free Full Text]
  37. Qian K, Chen H, Wei Y, Hu J, Zhu G 2005 Differentiation of endometrial stromal cells in vitro: down-regulation of suppression of the cell cycle inhibitor p57 by HOXA10? Mol Hum Reprod 11:245–251[Abstract/Free Full Text]
  38. Yao MW, Lim H, Schust DJ, Choe SE, Farago A, Ding Y, Michaud S, Church GM, Maas RL 2003 Gene expression profiling reveals progesterone-mediated cell cycle and immunoregulatory roles of Hoxa-10 in the preimplantation uterus. Mol Endocrinol 17:610–627[Abstract/Free Full Text]
  39. Bjornstedt M, Xue J, Huang W, Akesson B, Holmgren A 1994 The thioredoxin and glutaredoxin systems are efficient electron donors to human plasma glutathione peroxidase. J Biol Chem 269:29382–29384[Abstract/Free Full Text]
  40. Tham DM, Whitin JC, Kim KK, Zhu SX, Cohen HJ 1998 Expression of extracellular glutathione peroxidase in human and mouse gastrointestinal tract. Am J Physiol 275:G1463–1471
  41. Burton GJ, Jauniaux E 2004 Placental oxidative stress: from miscarriage to preeclampsia. J Soc Gynecol Investig 11:342–352[CrossRef][Medline]
  42. Jauniaux E, Burton GJ 2005 Pathophysiology of histological changes in early pregnancy loss. Placenta 26:114–123[CrossRef][Medline]
  43. Jauniaux E, Hempstock J, Teng C, Battaglia FC, Burton GJ 2005 Polyol concentrations in the fluid compartments of the human conceptus during the first trimester of pregnancy: maintenance of redox potential in a low oxygen environment. J Clin Endocrinol Metab 90:1171–1175[Abstract/Free Full Text]
  44. Apparao KB, Murray MJ, Fritz MA, Meyer WR, Chambers AF, Truong PR, Lessey BA 2001 Osteopontin and its receptor {alpha}vß(3) integrin are coexpressed in the human endometrium during the menstrual cycle but regulated differentially. J Clin Endocrinol Metab 86:4991–5000[Abstract/Free Full Text]
  45. Brown TL, Moulton BC, Witte DP, Swertfeger DK, Harmony JA 1996 Apolipoprotein J/clusterin expression defines distinct stages of blastocyst implantation in the mouse uterus. Biol Reprod 55:740–747[Abstract]
  46. Henriquez S, Tapia A, Quezada M, Vargas M, Cardenas H, Rios M, Salvatierra AM, Croxatto H, Orihuela P, Zegers-Hochschild F, Munroe DJ, Velasquez L 2006 Deficient expression of monoamine oxidase A in the endometrium is associated with implantation failure in women participating as recipients in oocyte donation. Mol Hum Reprod 12:749–754[Abstract/Free Full Text]
  47. Sherr CJ, Roberts JM 2004 Living with or without cyclins and cyclin-dependent kinases. Genes Dev 18:2699–2711[Abstract/Free Full Text]
  48. Steinman RA 2002 Cell cycle regulators and hematopoiesis. Oncogene 21:3403–3413[CrossRef][Medline]
  49. Kuiperij HB, van der Horst A, Raaijmakers J, Weijzen S, Medema RH, Bos JL, Burgering BM, Zwartkruis FJ 2005 Activation of FoxO transcription factors contributes to the antiproliferative effect of cAMP. Oncogene 24:2087–2095[CrossRef][Medline]
  50. Lynch RL, Konicek BW, McNulty AM, Hanna KR, Lewis JE, Neubauer BL, Graff JR 2005 The progression of LNCaP human prostate cancer cells to androgen independence involves decreased FOXO3a expression and reduced p27KIP1 promoter transactivation. Mol Cancer Res 3:163–169[Abstract/Free Full Text]
  51. Park Y, Maizels ET, Feiger ZJ, Alam H, Peters CA, Woodruff TK, Unterman TG, Lee EJ, Jameson JL, Hunzicker-Dunn M 2005 Induction of cyclin D2 in rat granulosa cells requires FSH-dependent relief from FOXO1 repression coupled with positive signals from Smad. J Biol Chem 280:9135–9148[Abstract/Free Full Text]
  52. Seoane J, Le HV, Shen L, Anderson SA, Massague J 2004 Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell 117:211–223[CrossRef][Medline]
  53. Risinger JI, Maxwell GL, Chandramouli GV, Jazaeri A, Aprelikova O, Patterson T, Berchuck A, Barrett JC 2003 Microarray analysis reveals distinct gene expression profiles among different histologic types of endometrial cancer. Cancer Res 63:6–11[Abstract/Free Full Text]
  54. Shazand K, Baban S, Prive C, Malette B, Croteau P, Lagace M, Racine JB, Hugo P 2004 FOXO1 and c-jun transcription factors mRNA are modulated in endometriosis. Mol Hum Reprod 10:871–877[Abstract/Free Full Text]
  55. Arden KC 2004 FoxO: linking new signaling pathways. Mol Cell 14:416–418[CrossRef][Medline]
  56. Essers MA, de Vries-Smits LM, Barker N, Polderman PE, Burgering BM, Korswagen HC 2005 Functional interaction between ß-catenin and FOXO in oxidative stress signaling. Science 308:1181–1184[Abstract/Free Full Text]
  57. Brisken C, Heineman A, Chavarria T, Elenbaas B, Tan J, Dey SK, McMahon JA, McMahon AP, Weinberg RA 2000 Essential function of Wnt-4 in mammary gland development downstream of progesterone signaling. Genes Dev 14:650–654[Abstract/Free Full Text]
  58. Sekiya T, Adachi S, Kohu K, Yamada T, Higuchi O, Furukawa Y, Nakamura Y, Nakamura T, Tashiro K, Kuhara S, Ohwada S, Akiyama T 2004 Identification of BMP and activin membrane-bound inhibitor (BAMBI), an inhibitor of transforming growth factor-ß signaling, as a target of the ß-catenin pathway in colorectal tumor cells. J Biol Chem 279:6840–6846[Abstract/Free Full Text]
  59. Sekiya T, Oda T, Matsuura K, Akiyama T 2004 Transcriptional regulation of the TGF-ß pseudoreceptor BAMBI by TGF-ß signaling. Biochem Biophys Res Commun 320:680–684[CrossRef][Medline]
  60. Onichtchouk D, Chen YG, Dosch R, Gawantka V, Delius H, Massague J, Niehrs C 1999 Silencing of TGF-ß signalling by the pseudoreceptor BAMBI. Nature 401:480–485[CrossRef][Medline]
  61. Jacobsen BM, Schittone SA, Richer JK, Horwitz KB 2005 Progesterone-independent effects of human progesterone receptors (PRs) in estrogen receptor-positive breast cancer: PR isoform-specific gene regulation and tumor biology. Mol Endocrinol 19:574–587[Abstract/Free Full Text]
  62. Brosens JJ, Hayashi N, White JO 1999 Progesterone receptor regulates decidual prolactin expression in differentiating human endometrial stromal cells. Endocrinology 140:4809–4820[Abstract/Free Full Text]
  63. Christian M, Pohnke Y, Kempf R, Gellersen B, Brosens JJ 2002 Functional association of PR and CCAAT/enhancer-binding protein ß isoforms: promoter-dependent cooperation between PR-B and liver-enriched inhibitory protein, or liver-enriched activatory protein and PR-A in human endometrial stromal cells. Mol Endocrinol 16:141–154[Abstract/Free Full Text]
  64. Dudoit S, Gentleman RC, Quackenbush J 2003 Open source software for the analysis of microarray data. Biotechniques Suppl:45–51
  65. Wettenhall JM, Smyth GK 2004 limmaGUI: a graphical user interface for linear modeling of microarray data. Bioinformatics 20:3705–3706[Abstract/Free Full Text]
  66. Hosack DA, Dennis Jr G, Sherman BT, Lane HC, Lempicki RA 2003 Identifying biological themes within lists of genes with EASE. Genome Biol 4:R70
  67. Livak KJ, Schmittgen TD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(-{Delta}{Delta} C(T)) Method. Methods 25:402–408[CrossRef][Medline]
  68. Essafi A, Fernandez de Mattos S, Hassen YA, Soeiro I, Mufti GJ, Thomas NS, Medema RH, Lam EW 2005 Direct transcriptional regulation of Bim by FoxO3a mediates STI571-induced apoptosis in Bcr-Abl-expressing cells. Oncogene 24:2317–2329[CrossRef][Medline]

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