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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 10 5233-5244
Copyright © 2004 by The Endocrine Society

Wild-Type p53 Protein Is Up-Regulated upon Cyclic Adenosine Monophosphate-Induced Differentiation of Human Endometrial Stromal Cells

Yvonne Pohnke, Tanja Schneider-Merck, Jasmin Fahnenstich, Rita Kempf, Mark Christian, Karin Milde-Langosch, Jan J. Brosens and Birgit Gellersen

Institute for Hormone and Fertility Research (Y.P., J.F.), University of Hamburg, and Endokrinologikum Hamburg (T.S.-M., R.K., B.G.), 20251 Hamburg, Germany; Institute of Reproductive and Developmental Biology (M.C., J.J.B.), Imperial College London, Hammersmith Hospital, London W12 ONN, United Kingdom; and Department of Gynecopathology (K.M.-L.), Institute of Pathology, University Hospital of Hamburg, 20246 Hamburg, Germany

Address all correspondence and requests for reprints to: Birgit Gellersen, Ph.D., Endokrinologikum Hamburg, Falkenried 88, 20251 Hamburg, Germany. E-mail: gellersen{at}endokrinologikum.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Decidualization of the endometrial stromal compartment is critical for embryo implantation. Initiation of this differentiation process requires elevated intracellular cAMP levels. We now report a massive and sustained up-regulation of p53 tumor suppressor protein during cAMP-induced decidualization of cultured endometrial stromal cells. Nuclear accumulation of p53 was not accompanied by increased mRNA expression, suggesting stabilization of the protein as the underlying mechanism. Proteasomal degradation of p53 is known to be mediated by nuclear Mdm2. Nuclear translocation of Mdm2, in turn, is dependent on phosphorylation by protein kinase B/Akt (PKB/Akt). In cAMP-treated decidualized cells, p53 accumulation was associated with decreased nuclear Mdm2 and cytoplasmic PKB/Akt levels. Conversely, withdrawal of the decidualization stimulus resulted in morphological and biochemical dedifferentiation, disappearance of p53, but increased abundance of PKB/Akt. Furthermore, Western blot and immunohistochemical analyses of endometrial biopsies confirmed that p53 is expressed in vivo in the stromal compartment during the late secretory phase of the cycle. The observation that p53 protein expression is closely associated with decidual transformation indicates a novel role for this tumor suppressor in regulating human endometrial function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SUCCESSFUL IMPLANTATION REQUIRES adequate synchronized maturation of the endometrial compartments. In humans, the first signs of decidualization of the stromal compartment appear in the cells surrounding the spiral arteries around d 23 of a regular 28-d cycle. Whereas this process is dependent on progesterone in vivo, decidualization of human endometrial stromal cells (ESCs) in vitro is effectively triggered by stimulation of the protein kinase A (PKA) signal transduction pathway. Elevation of the intracellular cAMP level by treatment with relaxin, CRH, or cAMP analogs leads to the decidualized phenotype within 2 d, whereas progesterone alone induces decidual transformation only after 6–14 d but serves to enhance the effect of prostaglandin E2, CRH, gonadotropin free {alpha}-subunit, or cAMP analogs (1, 2, 3). Key markers of decidualization, in addition to the morphological transition from the elongated fibroblastoid to a larger and rounder cell shape, are the induction of IGF binding protein (IGFBP)-1 and prolactin (PRL) gene expression (3). The PRL gene in decidual cells is transcribed from the alternative decidual PRL (dPRL) promoter, situated 5.7 kb upstream of the pituitary PRL promoter (4, 5, 6) and is transcriptionally controlled by cAMP (5). The sustained induction of the dPRL promoter in response to activation of the PKA pathway depends on the region –332/–270 relative to the transcriptional start site (7) and involves CCAAT/enhancer-binding protein (C/EBP)ß, a member of the CCAAT/enhancer-binding protein family of basic region/leucine zipper transcription factors (8), which binds to two sites in this region and serves as a critical transducer of the PKA signal to the dPRL promoter (9).

The tumor suppressor protein p53 is a transcription factor that is present at extremely low levels in normal cells. In response to genotoxic stress, p53 rapidly accumulates due to stabilization of the protein and ultimately leads to cell cycle arrest and DNA repair or to induction of apoptosis in damaged cells (10, 11). The p53 protein has a strong acidic transactivation domain at its N terminus (amino acids 1–42), which interacts with constituents of the basal transcription machinery and the integrator molecule cAMP response element-binding protein-binding protein/p300 (11, 12, 13). The consensus element for p53 consists of two copies of the palindromic sequence 5'-PuPuPuC(A/T)(T/A)GPyPyPy-3' and is occupied by a p53 tetramer. p53 exerts its biological function as the cellular gatekeeper for growth and division by transactivating cell cycle genes such as p21WAF1, growth arrest and DNA damage-inducible gene 45, or cyclin G (10). The p21 protein is an inhibitor of cyclin-dependent kinases and inhibits both the G1 to S-phase and the G2 to mitosis transitions (14, 15, 16). Whereas p53 induces transcription of the proapoptotic Bax gene, it represses transcription of the survival gene Bcl-2 and numerous other genes (11). Although the exact mechanism of transcriptional repression by p53 is still controversial, it has recently been demonstrated that induction of p21WAF1 expression is essential for negative regulation of gene expression by p53 (17).

The low level of p53 in normal cells is largely due to rapid turnover of the protein. The mouse double minute-2 (Mdm2) oncoprotein is a key regulator of p53. Not only does it bind to the N terminus of p53 and thus blocks its transactivation potential, but also it targets p53 for degradation via the ubiquitin-proteasome pathway by acting as an E3 ubiquitin ligase (18, 19). The cellular levels of the two proteins are linked in a negative feedback loop in that one of two alternative promoters of the Mdm2 (or the human ortholog Hdm2) gene itself is a transcriptional target of p53 (20). Up-regulation of Mdm2 expression by p53 then ensures termination of the p53 signal (21). The p53-Mdm2 regulatory loop is further fine-tuned by an intricate cross-link with the protein kinase B (PKB)/Akt signaling pathway. Akt, when activated by growth factors/survival signals, phosphorylates cytoplasmic Mdm2, which is a prerequisite for nuclear translocation of Mdm2 and its ability to interact with and down-modulate p53 (22). Phosphorylation and activation of Akt is triggered by the lipid mediator phosphatidylinositol-3,4,5-trisphosphate (PIP3) that recruits PIP3-dependent kinases and their substrate Akt to the plasma membrane. This process is antagonized by the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) (23). PTEN dephosphorylates PIP3 and thus inhibits Akt signaling. As a consequence, Mdm2 is not phosphorylated and remains in the cytoplasm in which it is subject to enhanced degradation, whereas nuclear p53 is protected from the inhibitory actions of Mdm2 (24). In a positive feedback loop, the PTEN promoter is transcriptionally induced by p53 (25), and furthermore, p53 can down-regulate Akt protein levels by enhancing caspase-mediated cleavage (26). The localization and phosphatase activity of PTEN are not restricted to the cytoplasm; PTEN has also been found in the nucleus in a cell cycle-dependent fashion, and it has been shown to interact with p53 and enhance its transcriptional activity (27, 28, 29).

In addition to its important role in maintaining integrity of the genome, there is emerging evidence for a role of p53 in development and differentiation. Inhibition of p53 activity by dominant-negative peptides blocks differentiation of rat neurons and primary muscle and hemopoietic cells, and p53-deficient leukemic cell lines can be induced to differentiate by introduction of wild-type p53 (30, 31, 32).

When we devised a yeast one-hybrid screening approach to identify transcription factors relevant to endometrial stromal cell differentiation, using dPRL promoter region –332/–270 as a bait, we identified p53 as a candidate protein. In this report, we describe a close link between the differentiated status and sustained elevation of p53 protein levels in ESCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

Purified human ESCs were prepared from anonymized biopsy samples obtained from premenopausal women at the time of hysterectomy for benign gynecological disorders. Informed consent was obtained, and the study was approved by the local ethics committee. Primary cultures were prepared as previously described (5) and maintained in DMEM/Ham’s F-12, 10% steroid-depleted fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 µg/ml insulin, and 1 nM 17ß-estradiol. Cells of the first passage were used for transfections and extraction of nuclear and cytoplasmic protein and RNA. Stimulations were performed with 0.5 mM 8-bromoadenosine-cAMP (8-Br-cAMP; Biolog, Bremen, Germany) or 250 nM progesterone (Sigma, Deisenhofen, Germany) for the indicated time periods. The Saos-2 human osteosarcoma cell line (HTB-85; American Type Culture Collection, Manassas, VA) and COS-7 cells were maintained in DMEM/Ham’s F12, 10% fetal calf serum, and antibiotics as above.

Transient transfection, protein extraction, and RNA isolation

Transient transfections for luciferase reporter gene assays were performed by the calcium phosphate precipitation method as described previously in triplicates using 24-well plates (33). One microgram of reporter construct and 0.05 µg of each expression vector were used. Controls received equimolar amounts of empty expression vector, and total DNA was kept constant by addition of promoterless plasmid DNA. Medium was replaced 16 h later. Cell harvest was performed for ESCs 48 h and for Saos-2 cells 24 h after medium replacement. Luciferase activity was measured with the luciferase reagent kit (Promega, Mannheim, Germany). Transfections were repeated at least three times, and representative experiments are shown (means ± SD). Statistical analysis was done by ANOVA with post hoc Bonferroni’s multiple comparison test, using GraphPad Prism 3.0a software for Macintosh (GraphPad Inc., San Diego, CA). Nuclear and cytoplasmic protein extracts from cultured cells were prepared as described by Gellersen et al. (33) and Schreiber et al. (34) with minor modifications. For RNA isolation the method developed by Gough (35) was applied, using the cytosolic lysate generated during protein extraction as the source.

Processing of endometrial biopsies for Western blot analysis and immunohistochemistry

Endometrial biopsies for Western blot analysis were taken at the time of laparoscopy for infertility or pelvic pain exploration and snap-frozen. Informed consent was obtained before the procedure and the study was approved by the local ethics and research committee. The mean age of the participants was 31.5 yr (range 26–38 yr). All women had regular menstrual cycles (mean 28.8 d; range 26–32 d) and a normal endometrial cavity on hysteroscopic inspection. Total protein was extracted in high salt buffer [20 mM HEPES (pH 7.4), 0.4 M KCl, 1 mM dithiothreitol, and 20% glycerol] containing a cocktail of protease inhibitors (0.5 mg/ml bacitracin, 40 mg/ml phenylmethylsulfonylfluoride, 5 mg/ml pepstatin A, 5 mg/ml leupeptin).

For immunohistochemical studies, tissue samples were obtained from five normal cycling women of reproductive age (mean age 39.2 yr; range 36–44 yr) between d 24 and 26 of the menstrual cycle. Snap-frozen specimens were fixed in a 3.5% buffered formaldehyde solution and embedded in paraffin. Serial sections of 4–6 µm were cut from paraffin blocks with normal endometrial tissue and mounted on aminopropyltriethoxysilane-coated slides, deparaffinized in xylene, and rehydrated in graded alcohol. For antigen retrieval, the slides were microwaved for 20 min in citrate buffer (pH 6.0) (ChemMate buffer for antigen retrieval, Dako, Glostrup, Denmark). Immunohistochemistry was performed in an automated system (Autostainer; Dako) with the ChemMate peroxidase/DAB detection kit (Dako). Rabbit polyclonal antibody to p53 (CM1; Novocastra Laboratories, Newcastle upon Tyne, UK) was used at a dilution of 1:100, monoclonal antibody to p53 (DO-1, 0.1 mg/ml; Oncogene, San Diego, CA) was used at a dilution of 1:2000. Rabbit polyclonal antibody developed against recombinant human wild-type p53 (SAPU, Carluke, Scotland, UK) was kindly provided by Dr. Wolfgang Bohn (Heinrich-Pette-Institute, University of Hamburg) and employed at a dilution of 1:8000. Sections were counterstained with hematoxylin. For negative controls, the primary antibody was omitted and replaced by the corresponding IgG isotype of the respective antibody.

All investigations described in this paper were conducted in accordance with the guidelines proposed in the Declaration of Helsinki (http://www.wma.net).

SDS-PAGE, Western blotting, and immunodetection

Proteins were electrophoresed on 10–12% SDS-polyacrylamide gels and transferred onto polyvinyl difluoride Immobilon membranes (Millipore, Eschborn, Germany). Immunodetection was performed with the enhanced chemiluminescence system (Pierce, Bonn, Germany). Rabbit polyclonal antibody against human p53 (FL-393; 2 mg/ml) and monoclonal Mdm2 antibody (SMP14; 200 µg/ml) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit antisera against human Bcl-2 and Bax and monoclonal p21WAF1 antibody (SXM30; 500 µg/ml) were from BD Biosciences PharMingen (San Diego, CA). Rabbit antibodies against total levels of Akt (recognizing Akt1, Akt2, and Akt3), phospho-Akt (Ser-473), and PTEN were from Cell Signaling Technology (Beverly, MA), and monoclonal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (clone 6C5) was purchased from HyTest (Turku, Finland). The following dilutions were used for Western blot analysis: p53 antiserum 1:5000, Bax antiserum 1:1000, Bcl-2 antiserum 1:500, PTEN and Akt antisera 1:1000, Mdm2 antibody 1:200, p21 antibody 1:500, and GAPDH antibody 1:5000. Secondary antibodies (horseradish peroxidase-conjugated antirabbit or antimouse IgG; Sigma) were employed at a dilution of 1:1000.

Immunofluorescence

Human ESCs of the first passage were plated in 24-well plates and treated with 0.5 mM 8-Br-cAMP for 6 d or left untreated. Monolayers were fixed with MetOH for 10 min at –20 C, washed with PBS, and permeabilized with 0.2% Triton X-100 for 10 min at room temperature. After washing with PBS, nonspecific binding was blocked with normal goat serum. Monoclonal antibody to p53 PAb421 (Oncogene) was diluted to 10 µg/ml in PBS. After 1 h incubation with primary antibody, cells were washed with PBS and incubated with Cy3-conjugated goat antimouse IgG (H+L) for 1 h (1:100 in PBS/2% normal goat serum) (Jackson ImmunoResearch, West Grove, PA). For nuclear counterstain, cells were incubated with 4,6-diamidino-2'-phenylindole (Sigma) at 1 µg/ml in PBS for 30 min. Cells were visualized with a fluorescence microscope (Epiphot, Nikon, Tokyo, Japan).

RT-PCR analysis

RNA was isolated from cytoplasmic lysates and used for oligo(dT)-primed cDNA synthesis with Ready-To-Go PCR beads (Amersham Biosciences, Freiburg, Germany). Semiquantitative PCR was carried out with the primer pairs listed in Table 1Go, and amplicons were separated on 1.2% agarose gels. After transfer to positively charged nylon membrane (Roche Applied Science, Mannheim, Germany), Southern blot hybridization was performed with internal oligonucleotides (Table 1Go) labeled with terminal deoxynucleotidyl transferase and digoxigenin-11-deoxyuridine 5-triphosphate, and visualized with the digoxigenin luminescent detection kit (Roche). Alternatively, PCR products were separated on 2% agarose gels and directly visualized by staining with SYBR Gold (Molecular Probes, Leiden, The Netherlands) and detection in a Typhoon 8600 imager (Amersham Biosciences).


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  		TABLE 1. Oligonucleotides used as primers and probes for RT-PCR analysis

 
Reporter constructs and expression vectors

The dPRL promoter/luciferase reporter fusion construct dPRL-332/luc3 and the expression vector for the long activating form of human C/EBPß, liver-enriched activator protein (LAP), in pSG5 have been described previously (9). The LAP insert was excised from pSG/LAP with EcoRI and BglII and inserted into the EcoRI and BamHI sites of pcDNA3.1(–) (Invitrogen, Karlsruhe, Germany) to yield pcDNA/LAP. Human p53 cDNA was amplified by RT-PCR on RNA from ESCs that had been treated with 8-Br-cAMP for 6 d. The sense primer introduced a HindIII site (underlined) by point mutation (lowercase) 44 bp upstream of the ATG start codon (5'-TTCCACGACGGTGACAaGCTTC-3') and the antisense primer an EcoRV site (underlined) by two point mutations (lowercase) 26 bp 3' to the termination codon (5'-GTGGGAGGaTaTCAGTGGGGAACA-3'), respectively. The PCR product was restricted with HindIII and EcoRV and inserted into pcDNA3.1(+) (Invitrogen), resulting in the eukaryotic expression vector pcDNA/hp53. Sequencing confirmed wild-type status of the hp53 insert. A 3' FLAG epitope was inserted as follows: pcDNA/hp53 was digested with Eco0109I just 5' to the stop codon and ApaI in the 3'-polylinker of pcDNA3.1(+). The resulting fragment was removed and replaced by a double-stranded oligonucleotide with overhangs to restore the 5'-Eco0109I and 3'-ApaI sites (italics) and including the FLAG epitope DYKDDDDK and a stop codon followed by a BamHI site (underlined): sense sequence; 5'-GGCCTGACTACAAAGACGATGACGACAAATAAGGATCCGGGCC-3', antisense sequence; 5'-GGGATCCTTATTTGTCGTCATCGTCTTTGTAGTCAG-3'. The resulting vector encodes hp53 amino acids 1–390 and was designated pcDNA/hp53/FLAG.

To generate pVP16-AD/hp53{Delta}TAD, the transactivation domain of hp53 was replaced by the VP16 transactivation domain. A sense oligonucleotide that anneals to hp53 cDNA at the codons for amino acids 75–81 and carries a 5'-overhang (italics) with an XhoI site (underlined) was used:

5'-TTTAAACTCGAGCCTGCACCAGCAGCTCCTAC-3'. This primer was paired with an antisense primer anchored in pcDNA3.1 downstream of the polylinker to perform PCR on template pcDNA/hp53/FLAG. The PCR product was restricted with XhoI and BamHI (immediately 3' to the FLAG sequence) and inserted into the respective sites of pABVP16. This vector was kindly provided by Drs. Sergio Onate and Sophia Tsai (Baylor College of Medicine, Houston, TX) and contains the coding region for the transactivation domain of Herpes simplex virus VP16 under control of the Rous sarcoma virus promoter.

For generation of yeast reporter plasmids, fragment dPRL–332/–270 was amplified using dPRL–332/luc3 as the template and primers with BamHI overhangs (underlined): 5'-AGGATCCATTATGTTCTGAGGGCTG-3' (sense) spanning the dPRL sequence –332 to –315, and 5'-AGGATCCGAGCAGAGACCAGACATG-3' (antisense), corresponding to positions –270 to –287, relative to the decidua-specific transcriptional start site. The PCR products were digested with BamHI, concatemerized, and cloned into pBM2389 (provided by Dr. D. Gietz, University of Manitoba, Winnipeg, Canada). An insert with three head-to-tail copies was excised by digestion with EcoRI and ligated in 5'-3' orientation into the EcoRI sites of pHISi or pLacZi (BD Biosciences Clontech, Palo Alto, CA) to yield the constructs 3x(dPRL–332/–270)/HISi and 3x(dPRL–332/–270)/LacZi, respectively. Control activator plasmids pGAD/cAMP response element modulator (CREM)-{alpha} and pGAD/PLZF-8ZF were constructed by inserting the DNA-binding domains of cAMP response element modulator-{alpha} and of promyelocytic leukemia zinc finger protein, respectively, into the yeast activator plasmid pGAD424 (BD Biosciences Clontech).

EMSA

A p53 consensus binding site (p53-RE) (Santa Cruz Biotechnology) was used as probe for EMSA. The double-stranded oligonucleotide was end labeled with [{gamma}-32P]ATP. Per binding reaction 5 µg of nuclear protein and 30,000 cpm of probe were used in the following binding buffer (Bf-B-high DTT): 20 mM Tris (pH 8.0), 1 mM EDTA, 50 mM dithiothreitol, 2 mM MgCl2, 0.1% Nonidet P-40, 10% glycerol, BSA 50 mg/ml, 0.02 U poly(dI-dC). After addition of probe, incubation was continued for 40 min at 4 C. For supershift analysis, 1 µl monoclonal antibody against p53 (clone PAb421; 1 mg/ml) (Oncogene) was added for an additional 20 min incubation at room temperature. Protein/DNA complexes were resolved on 4% nondenaturing polyacrylamide gels. Dried gels were exposed to x-ray film overnight at –80 C with intensifying screen.

Yeast one-hybrid system

Plasmids 3x(dPRL–332/–270)/HISi and 3x(dPRL–332/–270)/LacZi were integrated into the HIS3 or URA3loci, respectively, of the yeast strain YM4271 (BD Biosciences Clontech) to yield the reporter strains YM-3x332/270-HIS and YM-3x332/270-LacZ, respectively. Two reporter plasmids carrying p53 cis-acting sequences, p53HIS and p53BLUE (p53LacZ), provided with the Matchmaker one-hybrid kit (BD Biosciences Clontech), were also integrated into the YM4271 genome to give reporter strains YM-p53HIS and YM-p53LacZ, respectively. For activation, the positive control expression plasmid pGAD53m (mouse p53 cDNA in pGAD424) was used (BD Biosciences Clontech). Transformations were carried out using polyethylene glycol/lithium acetate, and HIS3 reporter strains were grown on HIS-selective medium in the presence of 15 mM 3-aminotriazol. The ß-galactosidase assay on LacZ reporter strains was carried out according to the supplier’s manual (BD Biosciences Clontech).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
p53 interacts with the dPRL promoter region –332/–270 in yeast and mammalian one-hybrid assays

In an attempt to identify transcription factors involved in differentiation of ESCs, we devised a strategy to use promoter fragment dPRL–332/–270 as a bait in a yeast one-hybrid system to isolate interacting proteins. Three copies were inserted in front of the His3 or LacZ reporter genes and integrated into the genome of yeast strain YM4271 to yield the reporter strains YM-3x332/270-HIS and YM-3x332/270-LacZ, respectively (Fig. 1Go). In the course of setting up the system, we performed positive control experiments by transforming the activator plasmid pGAD53m, which encodes a fusion of the GAL4 activation domain with mouse p53 (amino acids 72–390), into the p53-responsive reporter strains YM-p53HIS and YM-p53LacZ (Fig. 1Go). As negative controls, transformations of the same activator plasmid into the dPRL promoter/reporter strains YM-3x332/270-HIS and YM-3x332/270-LacZ were included. Unexpectedly, pGAD53m not only caused colony formation of YM-p53HIS but also of YM-3x332/270-HIS on His-selective media. Likewise, strong X-Gal staining was obtained not only of YM-p53LacZ but also of YM-3x332/270-LacZ colonies in the presence of pGAD53m. Transformations with the empty vector pGAD424, encoding the GAL4 activation domain only, or with pGAD/CREM-{alpha} or pGAD/PLZF-8ZF, encoding fusions of the GAL4 activation domain with the DNA-binding domains of two unrelated transcription factors, had no such effect (data not shown). These observations indicated binding of mouse p53 to dPRL–332/–270.



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  		FIG. 1. Schematic maps of reporter gene constructs and expression vectors. A, Reporter gene constructs for transfection of mammalian cells carried dPRL promoter sequence, exon 1a (black square) and the luciferase reporter gene (luc). Yeast reporter gene constructs harbored the HIS3 or LacZ reporter genes driven by the minimal GAL1 promoter. The C/EBP binding sites D and B of the dPRL promoter are indicated by gray ovals, and p53 response elements by black triangles. B, The mammalian expression vector for human p53, pcDNA/hp53, contains the full-length hp53 cDNA encoding a protein of 393 amino acids with transactivation domain (TAD), DNA-binding domain (DBD), and tetramerization domain (T4). In construct pVP16-AD/hp53{Delta}TAD, a 3' FLAG epitope was added and the homologous TAD replaced by the VP16 activation domain. The yeast activator construct pGAD53m, employed in the yeast one-hybrid assay, contains the corresponding region of the mouse p53 cDNA (residues 72–390) fused to the GAL4 activation domain in pGAD424.

 
To confirm an interaction of p53 with the dPRL promoter, a mammalian one-hybrid assay was carried out. To this end, a fusion construct encoding the VP16 activation domain linked to human p53 (amino acids 75–390; deletion of the transactivation domain), pVP16-AD/hp53{Delta}TAD, was generated (Fig. 1BGo) and cotransfected with the dPRL promoter/luciferase reporter construct 1x(dPRL–332/–270)/–32/luc3 (Fig. 1AGo), using the p53-deficient Saos-2 cell line. The VP16-AD/hp53{Delta}TAD fusion strongly activated reporter gene expression (Fig. 2AGo). Moreover, wild-type full-length hp53 transactivated as efficiently as the fusion protein. Coexpression of LAP was included for comparison because we reported previously that this isoform of C/EBPß has two binding sites in the dPRL–332/–270 region and activates the dPRL promoter (9).



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  		FIG. 2. Effect of p53 on dPRL promoter constructs in mammalian cells. A, The p53-deficient Saos-2 cell line was cotransfected with 1x(dPRL–332/–270)/–32/luc3 and the expression vectors pVP16-AD/hp53{Delta}TAD, pcDNA/hp53, pcDNA/LAP, or an equimolar amount of empty expression vector (control). B, Human ESCs were cotransfected with reporter construct dPRL–332/luc3 and expression vectors pcDNA/hp53, pcDNA/LAP, or empty expression vector (control). Luciferase activities are shown as relative light units (RLU; means ± SD of a representative experiment). *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with the respective controls. The values for LAP vs. hp53+LAP are significantly different (P < 0.001), whereas the values for hp53 vs. hp53+LAP are not.

 
p53 represses C/EBPß-dependent transactivation of the intact dPRL promoter

The reporter constructs used for the yeast and mammalian one-hybrid assays both presented the dPRL promoter element –332/–270 in a location immediately adjacent to the minimal promoter driving reporter gene expression (Fig. 1AGo). We now wanted to assess whether p53 would transactivate when the element –332/–270 was present in its natural context and distance from the transcriptional start site. The full-length promoter construct dPRL-332/luc3 (Fig. 1AGo) was therefore used for cotransfection analysis in primary cultures of ESCs (Fig. 2BGo). In contrast to the previous experiment, addition of hp53 resulted in a marked reduction of basal reporter gene activity. Moreover, p53 completely abolished LAP-induced activation. This was also observed in Saos-2 cells (data not shown) and was indicative of direct or indirect interaction between p53 and LAP, which blocks the DNA-binding and/or transactivation function of LAP.

p53 protein is up-regulated in decidualized ESCs

To determine whether p53-C/EBPß cross-talk may impact endometrial stromal cell function, we assessed the endogenous level of p53 in these cells. Two individual ESC preparations were treated for 6 d with 8-Br-cAMP, progesterone, or a combination of both or were left untreated. Nuclear proteins were harvested for Western blot analysis with p53 antibody (Fig. 3AGo). In untreated cells or cells treated with progesterone only, p53 was barely detectable. However, cAMP-treated cells displayed a massive induction of p53 protein independently of the presence of progesterone. cAMP-dependent accumulation of p53 was confirmed in 12 of 13 individual ESC preparations tested. Nuclear extracts from untreated and cAMP-treated cells were then compared by EMSA supershift analysis, using a p53 consensus binding site as the probe (Fig. 3BGo). Nuclear proteins from COS-7 cells, a Simian virus 40-transformed cell line that constitutively expresses wild-type p53 (36), were employed as a positive control and produced a strong signal that was supershifted on addition of monoclonal p53 antibody PAb421. In contrast, nuclear extracts from ESCs did not show binding to the p53 response element in the absence of antibody. However, when PAb421 was added, a supershift complex was formed specifically in extracts from cAMP-treated cells, irrespective of progesterone treatment (lanes 6 and 11). This indicates that the p53 protein, up-regulated in cAMP-treated cells, exists in a latent state but can be activated to bind DNA by PAb421. The ability of this particular antibody to enhance sequence-specific in vitro binding of p53 is well established (37). To investigate the kinetics of p53 up-regulation, ESCs were treated with 8-Br-cAMP for various periods of time, ranging from 1 h to 6 d. Nuclear and cytoplasmic proteins were then subjected to Western blotting (Fig. 3CGo). The accumulation of p53 in the nuclear compartment was not acute but set in only between 2 and 4 d of treatment and remained high thereafter. Cytosolic p53 was barely detectable; a faint, more slowly migrating doublet seen after 2 h might represent ubiquitinated p53 (lane 4).



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  		FIG. 3. p53 protein accumulates in ESCs on 8-Br-cAMP treatment. A, Two individual endometrial stromal cell preparations (ESC 1 and 2) were treated for 6 d with 8-Br-cAMP, progesterone (P), or a combination of both or left untreated. Western blot analysis of nuclear proteins (22.5 µg/lane) was performed with polyclonal antibody to p53. B, Nuclear proteins were harvested from ESCs treated as above or COS-7 cells and incubated with p53-RE as the probe for EMSA. For supershift analysis, monoclonal p53 antibody PAb421 was added. Lane 7 contained the free probe. S, Specific complex; SS, supershifted complex. The band migrating in the middle of the gel is a nonspecific complex (lanes 3–6, 8–11). C, ESCs were treated with 8-Br-cAMP for the indicated time periods (lanes 3–10) or left untreated (lane 2). Western blot analysis of nuclear proteins (15 µg/lane; upper panel) and cytosolic proteins (20 µg/lane; lower panel) was performed with polyclonal antibody to p53. Nuclear extracts from COS-7 cells were included as controls (lane 1).

 
By indirect immunofluorescence, we assessed the subcellular localization of p53 in ESCs with monoclonal antibody PAb421 that is directed against a C-terminal epitope (amino acid residues 371–380). In undifferentiated ESCs, p53 was only faintly detectable in the cytosol of some cells (Fig. 4Go). Intense speckled nuclear immunostaining was observed in most cells after 6 d of treatment with 8-Br-cAMP.



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  		FIG. 4. p53 protein localizes to the nuclei of ESC on 8-Br-cAMP treatment. ESCs were left untreated (–cAMP) or treated with 8-Br-cAMP for 6 d (+cAMP) and subjected to indirect immunofluorescence with p53 antibody PAb421 and Cy3 (Indocarbocyanine)-conjugated secondary antibody (left column). Nuclei were counterstained with 4,6-diamidino-2'-phenylindole (DAPI) (center column). Overlay (right column) shows nuclear localization of p53 in cAMP-treated cells (magnification: two upper panels, x20; bottom panel, x40). The scale bar (100 µm) applies to the two upper panels.

 
Decidualization and p53 accumulation are linked in cultured ESCs

To gain insight into the role and regulation of p53 in differentiating ESCs, we analyzed the expression profiles of a variety of p53 target genes and genes known to be involved in the regulation of p53 stabilization and decidual transformation. An ESC preparation was split into five parallel flasks and subjected to the following culture regimens: cultures a and b were maintained for 6 d either in the absence (a, –) or presence of 8-Br-cAMP (b, +). Cultures c and d were maintained for 12 d in the absence (c, –) or presence of 8-Br-cAMP (d, +), and culture e received 8-Br-cAMP for 6 d followed by 6 d of withdrawal (e, +/–). Cultures c–e were photographed after 9 and 12 d (Fig. 5AGo). The micrographs clearly show the fibroblast-like phenotype of proliferative untreated cells (culture c) and the fully decidualized rounded appearance of cAMP-treated cells (culture d) after 9 and 12 d. Cells that had been treated with cAMP for 6 d, followed by 3 d of withdrawal (culture e, 6d+/3d–, photographed at d 9), still appeared decidualized but had completely reverted to the fibroblastoid shape after 6 d of withdrawal (culture e, 6d+/6d–, photographed at d 12). Cultures a and b displayed the same morphological features as cultures c and d, respectively (not shown), i.e. full morphological decidualization was obtained within 6 d of cAMP treatment. Protein and RNA were extracted from a and b after 6 d and from c–e after 12 d in culture for further analysis.



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  		FIG. 5. cAMP-induced decidualization and p53 up-regulation in ESCs are reversible. Five parallel flasks (a–e) were plated from an ESC preparation and cultured as follows before extraction of nuclear (nuc) and cytoplasmic (cyt) proteins and cytoplasmic RNA: 6 d without stimulation (culture a), 6 d with 8-Br-cAMP (culture b), 12 d without stimulation (culture c), 12 d with 8-Br-cAMP (culture d), 6 d with 8-Br-cAMP followed by 6 d without stimulation (culture e). A, Cultures c–e were photographed after 9 and 12 d of treatment; + indicates presence, – indicates absence of 8-Br-cAMP (phase contrast; magnification, x20). B, Nuclear proteins (20 µg/lane) were subjected to Western blot analysis with antibodies against p53 (FL-393), PTEN, Mdm2, or p21WAF1. Cytoplasmic proteins (15 µg/lane) were subjected to Western blot analysis with antibodies against Bax, Bcl-2, total Akt, phospho-Akt (Ser-473), PTEN, or GAPDH. C, RT-PCR was performed to amplify fragments of the indicated mRNAs. Upper panel, Amplicons of dPRL, ICER, and GAPDH were subjected to Southern blotting and hybridized with internal oligonucleotide probes (Table 1Go). Lower panel, Amplicons of IGFBP-1, p53, S-Mdm2 (a product of the p53-inducible intronic promoter P2), L-Mdm2 (a product of promoter P1), PTEN, p21WAF1, Bax, and GAPDH were visualized by SYBR-Gold staining.

 
Nuclear proteins from all five cultures were subjected to Western blot analysis to assess their p53 status and the level of known p53 targets, Mdm2 and p21WAF1. The levels of Bax and Bcl-2, two additional well-characterized p53 target genes, were assayed in the cytoplasmic fraction. To address the involvement of Akt signaling in regulation of p53, we assessed Akt levels in the cytoplasm and PTEN levels in the cytoplasmic and nuclear fractions (Fig. 5BGo). As seen before, p53 was virtually undetectable in untreated cells but was markedly up-regulated after 6 d of cAMP-treatment (culture a vs. b). The p53 level was still elevated, albeit to a lesser extent, after 12 d of stimulation (culture c vs. d) but had returned to baseline after 6 d withdrawal of 8-Br-cAMP (culture e). The Mdm2 oncoprotein is commonly observed to migrate around 80 kDa and can be accompanied by a 60-kDa supposed cleavage product or, at least in tumor cells, by intermediate proteins arising from alternatively spliced transcripts (38, 39). We detected a higher abundance of Mdm2, migrating as a doublet between 80 and 60 kDa, in the nuclei of untreated compared with 8-Br-cAMP-treated cells. In 8-Br-cAMP-treated cells, only the higher-molecular-weight form was faintly expressed, whereas the smaller product reappeared in cells that had undergone 8-Br-cAMP stimulation followed by withdrawal.

The p21WAF1 cell cycle protein was readily detectable in all nuclear preparations and seemed to be only somewhat reduced in cells that had been subjected to 8-Br-cAMP withdrawal (e). The proapoptotic protein Bax was very abundant and, surprisingly, appeared to be inversely regulated to p53. Cells kept in the continuous presence of cAMP and displaying elevated p53 had lower levels of Bax. The antiapoptotic protein Bcl-2 showed a similar pattern; it was not detectable in cells that had significant levels of p53 (b, d). PTEN was less abundant in cAMP-treated cells in both cellular compartments; whereas cytoplasmic PTEN migrated at around 54 kDa as expected, an additional more slowly migrating band was detected in the nucleus the nature of which is unclear but which was regulated precisely in parallel with the 54-kDa species. Total Akt was also found to be down-regulated in cAMP-treated cells and returned to control levels on cAMP-withdrawal. Phospho-Akt was below the limit of detection after 6 d in culture but strongly up-regulated in untreated cells after 12 d. Addition of cAMP resulted in loss of phospho-Akt, whereas withdrawal of cAMP led to the reappearance of phospho-Akt. This indicates that cAMP negatively regulates the level of activated Akt.

RT-PCR analysis was then conducted to study the effects of cAMP on the transcript levels of p53 and its primary response genes p21WAF1, Bax, and Mdm2 (20, 40, 41). Two separate reactions were set up for amplification of Mdm2 mRNA to differentiate between products of the p53-inducible promoter P2 and the constitutive promoter P1. P1-driven transcription gives rise to L-Mdm2 mRNA, which includes leader exon 1, whereas P2-driven transcription generates S-Mdm2 mRNA including the alternative leader exon 2. The proteins translated from both transcript species are identical because the translational start codon is located in the common exon 3 (42, 43). The Bax gene can give rise to at least six different splice variants (44, 45, 46, 47). Because we primarily wanted to assess whether p53 in ESCs transactivates the Bax promoter, irrespective of the products, we chose the PCR primers such that they would amplify almost all potential variants (Table 1Go). Furthermore, we included PTEN mRNA in our analysis because the PTEN promoter has been reported to be responsive to p53, albeit with delayed kinetics, compared with the Mdm2 promoter (24). In addition, three marker transcripts of decidualization were amplified to correlate our findings to the differentiation status of the cells, namely dPRL; inducible cAMP early repressor (ICER), a cAMP-inducible product of the CREM gene (48); and IGFBP-1 (Fig. 5CGo). As expected, the dPRL promoter was silent in untreated cells (a, c) and massively induced after 6 and 12 d of incubation with 8-Br-cAMP, the level of mRNA being somewhat lower after 12 d (d vs. b). Interestingly, dPRL expression was almost completely lost from decidualized cells after 6 d of 8-Br-cAMP withdrawal (e) and thus correlated with the loss of morphological signs of decidualization in this population. A very similar pattern was obtained for IGFBP-1 and ICER mRNAs. Upon cAMP treatment, all four splice variants of ICER (ICER-I, -I{gamma}, -II, and -II{gamma}) were induced. We had previously reported that ICER mRNA undergoes a sustained up-regulation in decidualized cells (33) and now extend this finding by the observation that removal of the long-term cAMP stimulus results in down-regulation of ICER mRNA expression.

The p53, L-Mdm2, p21, and Bax mRNAs showed no cAMP-dependent regulation. PTEN mRNA was elevated after 12 d in culture, compared with 6 d, in the absence but not in the presence of 8-Br-cAMP.

The complete discordance between mRNA and protein levels observed for p53 indicates predominantly posttranscriptional regulation of p53 protein expression in ESCs. Likewise, the abundance of Mdm2 protein appears not to be determined by the rate of transcription. The level of S-Mdm2 mRNA was clearly induced in cAMP-treated cells, declined on withdrawal of cAMP, and correlated with p53 protein levels, consistent with p53-mediated induction of P2 of the Mdm2 gene.

Summarizing this experiment, 6 and 12 d of 8-Br-cAMP treatment elicited morphological decidualization, which coincided with up-regulation of p53 protein and dPRL, IGFBP-1, ICER, and S-Mdm2 mRNA expression; withdrawal of 8-Br-cAMP resulted in down-regulation of all five parameters and in morphological dedifferentiation. With the exception of S-Mdm2 mRNA, none of the targets of p53 was up-regulated either at the mRNA or protein level under conditions of elevated p53, whereas the protein level of Bcl-2, a factor known to be repressed by p53, was reduced. At the level of protein expression, Mdm2, Akt, and PTEN protein were coordinately down-regulated in the presence of cAMP.

p53 accumulates in vivo in the stromal compartment of secretory phase endometrium

To investigate whether up-regulation of p53 also occurs in vivo, we collected endometrial biopsies from different stages of the menstrual cycle. Four individual samples of each stage were analyzed by Western blotting (Fig. 6Go). p53 was low in the early proliferative phase, whereas biopsies from the mid- to late secretory phase (d 21–25) consistently showed the highest levels of p53. Five endometrial biopsies taken between d 24 and 26 of the menstrual cycle were subjected to immunohistochemical analysis to identify p53-positive cell type(s). This revealed strong staining for p53 in the majority of the nuclei in the stromal compartment, whereas the cytoplasm of stromal and epithelial cells and epithelial nuclei were only faintly positive (Fig. 7Go).



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  		FIG. 6. p53 protein is increased in the endometrium of the late secretory phase. Four endometrial biopsies each obtained in the early proliferative phase (d 4–8) (EP), late proliferative phase (d 9–14) (LP), early secretory phase (d 16–20) (ES), and mid- to late secretory phase (d 21–25) (LS) of the menstrual cycle, respectively, were analyzed for p53 expression. A total of 40 µg protein was loaded per lane on two parallel SDS gels (upper and lower panels) and immunodetected with polyclonal antibody to p53 (FL-393). COS-7 extracts were included for reference.

 


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  		FIG. 7. p53 is detectable in the endometrial stromal compartment in the secretory phase of the menstrual cycle. Paraffin sections from an endometrial biopsy taken in the late secretory phase of the cycle were subjected to immunohistochemistry with rabbit antibody to p53 (CM1) (A, magnification, x20; B, magnification, x40), a control section received normal rabbit IgG in place of primary antibody (C, magnification, 20x), and a hematoxylin/eosin (H&E) stain is shown in D (magnification, 10x). Strong positive p53 immunostaining (brown precipitate) is seen in nuclei of the stromal compartment. Counterstained p53-negative nuclei are blue. This case is representative of five cases studied. Similar results were obtained using a different polyclonal antibody (SAPU) and monoclonal p53 antibody DO-1. The scale bar in each panel represents 100 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
While searching for factors capable of regulating dPRL promoter activity, our attention was fortuitously drawn to p53 by its surprising ability to transactivate through the element dPRL–332/–270, when linked to a minimal promoter, in both a yeast one-hybrid and mammalian cotransfection systems. However, region –332/–270 does not contain p53 consensus binding sequences, and when it was presented in the natural context and distance from the transcriptional start site of the dPRL gene, p53 failed to transactivate. Likewise, p53 did not bind to this element in gel shift assays even in the presence of activating antibody PAb421 (data not shown). We therefore believe that –332/–270 contains very low affinity binding sites for p53 and that binding of p53 to these sites is stabilized only in intact cells because of their proximity to the transcriptional start site and the ability of p53 to interact with numerous components of the basal transcription machinery such as TATA-box binding protein, TATA-box binding protein-associated factors TAF-40 and TAF-60, and transcription factor TFIIH (11). These components do not assemble in vitro on the isolated –332/–270 fragment, leading to the failure of p53 to bind in EMSA. When the intact dPRL promoter was transfected, p53 even repressed its LAP-dependent activation. Such p53-mediated inhibition of C/EBPß-activated transcription has previously been reported for the albumin, IL-6, and insulin receptor gene promoters (49, 50, 51). However, the mechanism of this transcriptional antagonism remains to be elucidated.

p53 plays a fundamental role in maintaining integrity of the genome in normal cells. The tumor suppressor function of p53 relies on its rapid accumulation in the nucleus in response to genotoxic insults. This either triggers the irreversible process of apoptosis or arrests the cell cycle in G1 or G2 to allow for DNA repair. The stress-induced p53 signal is terminated by induction of Mdm2, which binds to p53, blocks its transactivation function, and initiates its proteasomal degradation (21). A chronically high level of p53 in cells of the adult organism is a phenomenon usually restricted to tumor cells that express mutant p53 protein that lacks transcriptional activity. As a consequence, the p53-responsive Mdm2 gene promoter is not induced, the negative feedback loop is disabled, and p53 stabilized (18). Here we report the novel observation of a sustained up-regulation of p53 in decidualized ESCs. The elevated levels of p53 were not associated with enhanced transcription of the p53 gene, suggesting stabilization of the protein as the underlying mechanism. Several lines of evidence support the notion that the accumulation of p53 in decidualized cells is not the consequence of mutation. First, the p53 cDNA was cloned from cAMP-treated ESCs, sequenced, and found to be wild type. Second, in all individual specimens tested, the p53 level in undifferentiated ESCs was low or undetectable, attesting to the wild-type status of the donors. It appears extremely unlikely that 8-Br-cAMP treatment would reproducibly cause a stabilizing mutation. Even so, such first-hit mutation would require an even more unlikely second hit to either mutate, delete, or silence the second allele because in a heterozygous situation, the expression of p53 from the wild-type allele would suffice to initiate the autoregulatory degradative pathway (52). Finally, up-regulation of p53 was completely reversible. Withdrawal of the decidualizing stimulus led to the loss of p53 protein expression, arguing against a genetic background of altered p53 status. Furthermore, the induction of p53 in our in vitro decidualization model reflected the in vivo situation because we demonstrated, for the first time, that p53 protein increases in normal endometrium with progression through the menstrual cycle.

Immunohistochemical studies on p53 expression in the normal endometrium are sparse. The focus has usually been placed on malignancies arising from the epithelial compartment, which are much more frequent than stroma-derived sarcomas. When progression from benign proliferative endometrium through endometrial hyperplasia to endometrial adenocarcinoma was studied, p53 was not detected in the first two stages. In endometrial carcinoma, about 50% of the cases showed nuclear staining for p53 in the epithelial cells, most likely as the result of mutation (53). A hormone-dependent increase in p53 protein under physiological conditions has been observed in pregnant-involuted mammary glands of rats and mice. The sustained up-regulation of nuclear p53 could be recapitulated by long-term exposure of nonpregnant animals to pregnancy levels of estrogen plus progesterone. Up-regulation of p53 in the mammary epithelium was, however, separable from differentiation per se and was proposed to be the basis for pregnancy- and hormone-induced resistance to mammary carcinogenesis in rodents (54).

The mechanism underlying the nuclear accumulation of p53 in differentiating endometrial stromal cells is not entirely clear. The most obvious yet paradoxical explanation for stabilization of p53 would be a concomitant down-regulation of Mdm2. We in fact observed a distinct decrease in nuclear Mdm2 protein after 6 and 12 d of 8-Br-cAMP treatment. Intriguingly, this occurred in the presence of unaltered L-Mdm2 transcript levels and increased abundance of S-Mdm2 transcripts. Whereas the upstream Mdm2 promoter P1 governs p53-independent transcription (42), P2 is p53-responsive, and the increase in S-Mdm2 mRNA might point to transcriptional activity of the up-regulated p53 protein. Use of P2 leads to the generation of the S-Mdm2 transcript species, which displays significantly elevated translational efficiency, compared with L-Mdm2 transcripts (42, 43) and should therefore yield increased Mdm2 protein in p53-expressing cells, contrary to what we observed in ESCs. However, reduced nuclear Mdm2 in the presence of increased Mdm2 transcript levels may result from a block in nuclear entry. Mdm2 must be phosphorylated by Akt to translocate to the nucleus (22). Hypophosphorylated Mdm2 is restricted to the cytoplasm and subject to degradation, thus accounting for stabilized p53. One way in which p53 can be protected from Mdm2 is the phosphatase activity of PTEN (24). PTEN antagonizes the effect of phosphatidylinositol 3-kinases, which convert PI(4,5)P2 to PIP3, which in turn activates Akt (55). The presence of PTEN has previously been demonstrated by immunohistochemistry in endometrial biopsies (56, 57). We investigated whether increased PTEN might account for the stabilization of p53 in decidualized cells, but Western blot analysis rather revealed a decrease in PTEN on cAMP-induced decidualization. Notably, however, we observed a decreased abundance of Akt protein, the kinase responsible for Mdm2 phosphorylation, in cAMP-treated cells, offering an explanation for accumulation of p53 protein.

In a different study, a massive increase in total Akt levels in ESCs has been observed with time in culture (58). This up-regulation occurred both in control cells and cells induced to decidualize by exposure to estradiol plus progesterone and thus was independent of the hormonal treatment. However, the steroids caused a distinct reduction in phosphorylated Akt, compared with control cells of the same time point. This reduction was reversed by treatment with the PKA inhibitor H89. In addition, 8-Br-cAMP treatment decreased the amount of phospho-Akt within 15 min, indicating that the cAMP/PKA pathway is a negative modulator of Akt activity in stromal cells (58). In corneal epithelial cells, 8-Br-cAMP has been shown to inhibit phosphatidylinositol 3-kinases and promote rapid turnover of Akt (59). In addition, cAMP, via stimulation of PKA, can cause reduced phosphorylation of Akt and thereby inhibit its activity (60). In our system, we observed both a reduction of total and of phospho-Akt levels in decidualized cells on long-term exposure to cAMP.

We propose that the decrease in Mdm2 protein, by an apparently posttranscriptional mechanism, in 8-Br-cAMP-treated ESCs at least in part accounts for accumulation of the p53 protein. Enhanced degradation and/or nuclear exclusion of Mdm2 may be a consequence of reduced Akt, as outlined above. Interestingly, another transcription factor intimately involved with cAMP-induced decidualization, Forkhead (FOXO)1a (formerly FKHR), is also subject to Akt-mediated regulation (3, 61). FOXO1a is a member of the FOXO family of forkhead transcription factors and is a target of Akt-induced phosphorylation. Phosphorylation inhibits transcriptional activity of FOXO1a by promoting relocalization from the nucleus to the cytoplasm (62). We demonstrated previously that in decidualized ESCs, both in culture and in vivo, FOXO1a is strongly induced and accumulates in the nucleus (61). This further supports our notion of a reduction in Akt on decidualization as described in this report. We can, however, not formally exclude that the loss of Akt is secondary to the increase in p53 because activated p53 has been shown to enhance caspase-mediated cleavage of Akt (63).

Whether p53 is transcriptionally active in decidualized ESCs is unclear. Although we observed increased transcript levels of S-Mdm2, the product of the p53-responsive P2 promoter, in p53-expressing ESC cultures, this is not conclusive evidence for transcriptional activity of p53. In fact, p53-independent activation of the Hdm2 P2 promoter in estrogen receptor-{alpha}-positive breast cancer cells has recently been described (64). Moreover, the kinetics of p53 accumulation in differentiating ESCs argue against an activated status of the protein. DNA-binding and transcriptional activity of p53 are believed to be induced by posttranslational modifications in response to acute stress (65). Such phosphorylation, acetylation, and redox events (66) are most likely acute and of transient nature, whereas the accumulation of p53 in cAMP-treated ESCs was found to set in with a delay of several days. The expression of several transcriptional targets of p53, namely the p21WAF1, Bax, and PTEN genes, was not increased by the persistently elevated p53 protein in decidualized ESCs.

It is therefore conceivable that p53 in decidualizing ESCs may function predominantly as a transcriptional repressor. It is of interest to note that we have previously observed other negative regulators of transcription to be up-regulated in the course of decidualization. Among these are ICER (Ref.33 and this report); the short inhibitory form of C/EBPß, LIP (9); and C/EBP{gamma}, a transdominant negative member of the C/EBP family (67) (Pohnke Y. and B. Gellersen, unpublished data). All of these factors, including p53, have in common that their cAMP-induced increase in ESCs is of a persistent nature and coincides with the onset of dPRL expression as a marker of decidualization. We propose that a fine balance between activators and repressors of transcription controls differentiation of the endometrial stroma and allows further proliferation of the tissue that is required to form the decidua of pregnancy. Interestingly, within the time frame studied here (up to 6 d), progesterone did not induce p53 accumulation. This correlates with the much weaker decidualization efficacy of progesterone, compared with cAMP.

In summary, our report adds further evidence to the concept that p53 not only serves to guard the cell against genotoxic insults but also participates in differentiation processes. Whether the accumulation of wild-type p53 in ESCs is a prerequisite for or a consequence of decidualization will be the subject of future studies.


   Acknowledgments
 
The authors are indebted to Dr. H. K. Pauli and Professor C. Lindner (Elim Hospital, Hamburg, Germany) for providing hysterectomized tissue and Drs. S. Gordts and R. Campo (Leuven Institute for Fertility and Embryology, Leuven, Belgium) and Dr. P. Puttemans (Department of Obstetrics and Gynaecology, St. Elisabeth Hospital, Brussels, Belgium) for collecting endometrial biopsies. We thank Gabriele Rieck for excellent technical assistance and Drs. D. Gietz, S. Tsai, S. Onate, and W. Bohn for plasmids and antibodies. We gratefully acknowledge the continuous support of Professor F. Leidenberger (Institute for Hormone and Fertility Research, Hamburg).


   Footnotes
 
This work was supported by the Deutsche Forschungsgemeinschaft (DFG Ge 748/1-4, Ge 748/9-1) (to B.G.), a Wellcome Trust Clinical Scientist Fellowship (54043) (to J.J.B), and a Royal Society Joint Project Grant.

Abbreviations: 8-Br-cAMP, 8-Bromoadenosine-cAMP; C/EBP, CCAAT/enhancer-binding protein; CREM, cAMP response element modulator; dPRL, decidual PRL; ESC, endometrial stromal cell; FOXO, Forkhead; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ICER, inducible cAMP early repressor; IGFBP, IGF binding protein; LAP, liver-enriched activator protein; Mdm2, mouse double minute-2; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PKA, protein kinase A; PKB, protein kinase B; PRL, prolactin; PTEN, phosphatase and tensin homolog deleted on chromosome 10.

Received January 6, 2004.

Accepted July 21, 2004.


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 Introduction
 Materials and Methods
 Results
 Discussion
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