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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-0769
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 4 1453-1461
Copyright © 2006 by The Endocrine Society

Dickkopf-1, an Inhibitor of Wnt Signaling, Is Regulated by Progesterone in Human Endometrial Stromal Cells

Suzana Tulac, Michael T. Overgaard, Amy E. Hamilton, Nelson L. Jumbe, Ernest Suchanek and Linda C. Giudice

Department of Obstetrics, Gynecology, and Reproductive Sciences (S.T., M.T.O., A.E.H., L.C.G.), University of California, San Francisco, San Francisco, California 94143-0132; Department of Pharmacokinetics, Pharmacodynamics, and Bioanalytical Sciences (N.L.J.), Genentech Inc., South San Francisco, California 94080; and Department of Obstetrics and Gynecology (E.S.), Zagreb University School of Medicine, Zagreb 10000, Croatia

Address all correspondence and requests for reprints to: Linda C. Giudice, M.D., Ph.D., M.Sc., Professor and Chair, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, 505 Parnassus Avenue M1496, San Francisco, California 94143-0132. E-mail: giudice{at}obgyn.ucsf.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Some members of the Wnt family, including ligands, receptors, inhibitors, and signaling components, are expressed in human endometrium. Dickkopf-1 (Dkk-1), a potent inhibitor of the Wnt signaling pathway, was recently found to be up-regulated in decidualizing endometrial stromal cells during the secretory phase of the menstrual cycle, suggesting regulation by progesterone.

Objectives: To test the hypothesis that progesterone regulates Dkk-1 expression in human endometrial stromal cells, we investigated the following effects on stromal cell expression of Dkk-1 mRNA and protein: decidualizing stimuli (progesterone or cAMP), RU486 (an inhibitor of progesterone action), and withdrawal of progesterone.

Results: Short-term treatment (up to 72 h, which corresponds to the full decidualized phenotype in response to cAMP and an early response to progesterone) did not reveal regulation of Dkk-1 mRNA or protein by cAMP but did show induction of Dkk-1 expression when the cells were treated with progesterone, an effect that was blocked by RU486. In long-term cultures (from 14 to 23 d, which corresponds to the full decidualized phenotype in response to progesterone), a significant increase in Dkk-1 mRNA and protein production was observed. Addition of RU486 or withdrawal of progesterone after long-term decidualization resulted in a decrease of Dkk-1 mRNA and protein to control levels. Estradiol alone had no effect on stromal Dkk-1 expression.

Conclusions: These data strongly support regulation by progesterone of Dkk-1 mRNA synthesis and protein expression in human endometrial stromal cells and that the response is specific for progesterone and independent of cAMP and estradiol.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE STEROID HORMONES estradiol (E2) and progesterone are key regulators of cellular proliferation, secretory protein production, and cellular differentiation in reproductive tissues including the endometrium (1). Progesterone also plays a pivotal role in ovulation, implantation, and establishment and maintenance of pregnancy. During the secretory phase, progesterone promotes endometrial stromal cell decidualization, associated with distinct morphological and functional features. With the development of in vitro models of decidualization of endometrial stromal cells (2, 3), studying the molecular mechanisms underlying decidualization has been enabled with prolactin (4, 5), IGF binding protein (IGFBP)-1 (6, 7), laminin, and fibronectin (8, 9) protein expression, and down-regulation of smooth muscle actin (10) considered to be molecular markers of the decidualization process, corresponding to events in vivo. Stromal cell decidualization can also be stimulated in vitro by 8-bromo cAMP or other stimuli that activate the protein kinase A-dependent pathway (3, 11), usually within a short time frame (up to 72 h), compared with the prolonged response to the progesterone (days to weeks) (12).

Previous global gene profiling studies from our laboratory (13) and others (14, 15, 16, 17) and validation and cellular localization studies (18) have demonstrated that some members of the Wnt family are expressed and regulated across the menstrual cycle in human endometrium, suggesting their possible regulation by steroid hormones. We previously demonstrated (18) that Dickkopf-1 (Dkk-1) and frizzled related protein HE (frpHE), inhibitors of Wnt signaling, are up-regulated and down-regulated, respectively, in normal human endometrium during the midsecretory phase (window of implantation) of the menstrual cycle, when circulating progesterone levels are at their peak.

Wnt signaling proteins comprise a family of molecules that exhibit pivotal roles in cell proliferation, differentiation and polarity, epithelial-mesenchymal communication, and embryogenesis (19, 20, 21). Signaling events are initiated by Wnt ligand binding to its cell surface receptor, frizzled. Ligand-receptor interaction activates disheveled protein, leading to inactivation of glycogen synthase kinase-3ß and cytoplasmic ß-catenin dephosphorylation and accumulation and its subsequent entry into the nucleus. Once activated, ß-catenin interacts with the DNA binding T cell factor and lymphoid enhancing factor-1, thereby regulating Wnt target genes such as c-myc, cyclin D1, matrilysin, peroxisome proliferator-activated receptor-{gamma}, cyclo-oxygenase-2, muscle segment homeobox 1, and others (22, 23, 24, 25, 26). Several families of inhibitors of Wnt signaling have been identified. Secreted frizzled related proteins share structural similarities to the frizzled receptor family of proteins and can antagonize Wnt action at the level of receptor-ligand binding (27, 28). Dkk-1 interacts with low-density lipoprotein receptor-related protein-5/6 and inhibits Wnt signaling by disrupting the binding of lipoprotein receptor-related protein-5/6 to the Wnt/Fz ligand-receptor complex (29, 30, 31). Dkk-1 functions during development (32, 33, 34), and it has been recently demonstrated that Dkk-1 is a proapoptotic gene that may play important roles in both oncogenic Wnt and p53 tumor suppressor pathways (29, 35).

The observation of cycle-dependent expression of Dkk-1 in human endometrium during the secretory, compared with the proliferative, phase and its cell-specific expression in endometrial stroma raises the question as to whether progesterone regulates Dkk-1 in this tissue. To this end we investigated regulation of Dkk-1 mRNA and protein expression in human endometrial stromal cells decidualized in vitro with progesterone and subsequently treated with RU486 (a progesterone receptor antagonist) or progesterone withdrawal. We also investigated regulation of Dkk-1 by 8-bromo cAMP, another decidualizing stimulus classically considered downstream from progesterone during decidualization (36, 37). The data support that progesterone, but not cAMP, is an activator of Dkk-1 expression in human endometrial stromal cells.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Tissue specimens

Endometrial samples were obtained from normally cycling women after written informed consent, under an approved protocol by the Stanford University Committee on the Use of Human Subjects in Medical Research (Stanford, CA) and the University of California, San Francisco Committee on Human Research. Some of the tissues were obtained from the NIH U54 UCSF/Stanford Tissue and DNA Bank, now based in San Francisco, California. Histologically normal endometrial tissue samples were obtained by biopsy or hysterectomy from 15 different subjects between 26 and 46 yr of age, who had regular menstrual cycles (28–35 d), were documented not to be pregnant, and had no history of endometriosis. Samples were collected at the room temperature and transported to the laboratory in DMEM (Life Technologies, Inc., Grand Island, NY) and processed for the endometrial stromal cell isolation as described below.

Cell cultures

Endometrial tissue was subjected to collagenase/hyaluronidase (Sigma-Aldrich, St. Louis, MO) digestion for 2 h at 37 C. After digestion, the stroma was dispersed, whereas the epithelial structures remained mostly intact. Human endometrial stromal cells were separated from epithelium on a size basis, as previously described (38) and were resuspended in a plating media consisting of a 4:1 ratio of DMEM-F12 (Life Technologies) and DMEM+10% fetal bovine serum (Life Technologies). The cells were preplated in 10 cm2 standard culture plates (Costar, Corning, NY) for 1 h at 37 C and 9% CO2. The plating medium was replaced after 1 h with maintenance medium consisting of high-glucose DMEM/MCDB-105 medium with 10% charcoal-stripped fetal bovine serum, insulin (5 µg/ml) (Sigma-Aldrich), gentamicin, penicillin, and streptomycin, as described (39). Endometrial stromal cells were passaged two to three times before the experiments. Purity of the stromal cell cultures was confirmed by vimentin immunostaining. Human endometrial stromal cells obtained from 10 different endometrial samples were used for total RNA isolation and further for quantitative RT-PCR, dot-blot, and Northern blot analyses. Five additional endometrial samples that were prepared for protein were used for stromal cell cultures analysis.

Treatment of endometrial stromal cells in vitro

All treatments were carried out in serum-free medium consisting of 75% DMEM, 25% MCDB-105, 50 µg/ml ascorbic acid, and 5 µg/ml transferrin.

Short-term cultures. Short-term cultures (up to 72 h) of human endometrial stromal cells were established in serum-free medium to investigate the effects of 8-bromo cAMP, estradiol, progesterone, and RU486 on Dkk-1 expression. This short time period corresponds to maximal decidualization in response to cAMP and an early response to progesterone, with regard to the decidual phenotype. Endometrial stromal cells were plated in 4 cm2 plates (Costar) and treated separately with 1 mM 8-bromo cAMP (Sigma-Aldrich); 1 µM progesterone + 10 nM estradiol (E2P4); 10 µM RU486 (Sigma-Aldrich) + E2P4; 10 nM E2 alone; 10 µM RU486 alone; and serum-free medium with addition of vehicle control (nondecidualized control) for the equivalent time in culture.

Long-term cultures. After 8–16 d of E2P4 treatment, endometrial stromal cells produce IGFBP-1, a decidual marker, in amounts that are comparable with 48–72 h of treatment with cAMP (12, 40, 41). Confluent endometrial stromal cells were decidualized in vitro with 1 µM progesterone + 10 nM estradiol (E2P4) for 17 d in serum-free medium. Stromal cells were also treated with 1 mM 8-bromo cAMP that, after 72 h of treatment, resulted in production of equivalent levels of IGFBP-1, compared with the long-term (17 d) treatment with E2P4. Human endometrial stromal cells cultured in the serum-free medium without additives, but with the addition of vehicle control for the equal period of time in culture, were used as nondecidualized controls. The concentration of IGFBP-1 in conditioned media (CM) was determined using an IGFBP-1 ELISA (Diagnostics Systems Laboratories, Webster, TX). After cells were decidualized for 14 d using serum-free media containing E2P4 (t = 0), they were separated into three groups: the first group was continuously treated with E2P4; the second group was treated with E2P4 with the addition of 10 µM RU486; and the third group was treated in serum-free media alone (E2P4 withdrawal) for 3, 6, and 9 additional days after t = 0. Cells were incubated at 37 C and 9% CO2, and the media were renewed every 3 d.

RNA extraction, reverse transcription, and quantitative PCR

Total RNA from endometrial stromal cells obtained from five different subjects was isolated and purified using RNeasy minikit (QIAGEN, Valencia, CA), following the manufacturer’s protocol. Total RNA (1 µg) was reverse-transcribed using Omniscript kit (QIAGEN) according to the manufacturer’s instructions with a 1:1 ratio of oligo (dT)16–18 and random hexamers (Invitrogen, Carlsbad, CA). Dkk-1 mRNA regulation in endometrial stromal cells treated with E2P4, E2P4 with addition of RU486 or withdrawal of E2P4 was measured by quantitative PCR using human Dkk-1 QuantiTect assay (QIAGEN). The QuantiProbe sequence for Dkk-1 was 5'-CACACCAAAGGACAAGA-3'. The forward primer sequence for Dkk-1 was 5'-GGGAATTACTGCAAAAATGGAATA-3', and the reverse primer sequence was 5'-ATGACCGGAGACAAACAGAAC-3'. QuantiTect HsRRN18S assay (QIAGEN) was used as a normalizer. Real-time PCR was performed in triplicate in 25-µl reactions using the QuantiTect probe PCR kit (QIAGEN), following the manufacturer’s instructions and carried out in the Mx4000 Q-PCR system (Stratagene, La Jolla, CA). The thermal cycling conditions included an initial activation step at 95 C for 15 min, followed by 40 cycles of denaturation, annealing and amplification (94 C for 15 sec, 56 C for 30 sec, 76 C for 30 sec, respectively). Fluorescence data collection was performed during the annealing step. For standard curve construction Dkk-1 and 18S RT-PCR products were cloned by A-T cloning into the pDrive cloning vector (QIAGEN). One nanogram of pDrive vector with cloned Dkk-1 or 18S gene expression assays corresponds to 2.4 x 108 copies of the respective target. Standard curves for Dkk-1 and 18S were obtained from six 10-fold dilutions (2.4 x 107, 2.4 x 106, 2.4 x 105, 2.4 x 104, 2.4 x 103, and 2.4 x 102). The efficiencies of amplification for both genes were calculated according to the equation: efficiencies of amplification = 10[–1/slope] – 1 and ranged from 101 to 103%. For each sample, the amount of Dkk-1 and normalizer (18S) was determined from their respective standard curves.

Northern and dot-blot analysis

Total RNA prepared from cultures obtained from four different subjects was used for dot-blot analysis and two for Northern blot analysis. Total RNA (10 µg for dot-blot and 20 µg for Northern blot) was denatured, electrophoresed on 1% formaldehyde agarose gel, and transferred to the membrane for Northern blot analysis or directly blotted onto membranes through the convertible filtration manifold system (Invitrogen) for dot-blot analysis. Specific radioactive probes for Dkk-1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were generated with the Ready-to-Go random primer kit (Pharmacia Biotech, Peapack, NJ) and 32{alpha}P-dCTP (NEN Life Science Products, Boston, MA), followed by a cleanup on the MicroSpin S-200 HR columns (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). Membranes were prehybridized at 68 C for 60 min in ExpressHyb buffer (Clontech, Palo Alto, CA) containing 100 µg/ml salmon sperm DNA (Invitrogen) and the hybridization carried out for another hour at 68 C using buffer containing 1–2 x 106 cpm/ml of labeled probe. After washing (according to the manufacturer’s instructions), membranes were exposed to MS x-ray films (Kodak, Rochester, NY), scanned in the GS-710 imaging densitometer (Bio-Rad Laboratories, Hercules, CA), and analyzed by its accompanied software Quantity One (Bio-Rad Laboratories, version 4.0.2). GAPDH mRNA intensities were used for normalization. Stripping and reprobing with GAPDH as a constitutively expressed marker were performed using the same membranes.

CM preparation and enzymatic deglycosylation

For each sample, 10 volumes of 96% ethanol were added to 100 µl CM, placed for 30 min in –20 C, and then centrifuged at 10,000 x g for 15 min. Pellets were dissolved in 20 µl Laemmli sample buffer, boiled for 5 min, chilled on ice, and loaded onto 4–20% SDS-polyacrylamide gels (Gradipore, Frenchs Forest, Australia). For enzymatic deglycosylation, prepared pellets were resuspended in 25 µl of 1x glycoprotein denaturing buffer containing 0.5% SDS and were boiled at 100 C for 10 min. After boiling, samples were incubated in 1x G7 reaction buffer supplemented with 1% Nonidet P-40 and N-glycosidase F (New England Biolabs, Inc., Beverly, MA) for 2 h at 37 C. The digests were analyzed by Western blotting using an anti-Dkk-1 antibody (R&D Systems, Inc., Minneapolis, MN). Glycosylated recombinant human Dkk-1 protein (R&D Systems), which migrates at approximately 37 kDa, was used as a control.

Western blot analysis

Endometrial stromal cells obtained from five different subjects were used for protein analysis. After the indicated treatment, stromal cells were washed with PBS, lysed in radioimmunoprecipitation assay lysis buffer (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), centrifuged for 10 min at 10,000 x g, and proteins were denatured by boiling for 5 min in Laemmli sample buffer (Bio-Rad). Protein quantity in the cell lysates was evaluated by the Bradford protein assay (Bio-Rad) following the manufacturer’s instructions. Twenty micrograms of total protein lysates per lane or CM (prepared as described previously) were loaded onto 10% SDS-polyacrylamide gels (Gradipore) for cell lysates or 4–20% SDS-polyacrylamide gels for CM and then transferred to nitrocellulose membranes (Schleicher & Schuell Bioscience, Dassel, Germany) by electroblotting. After transfer, membranes were placed in blocking buffer (5% nonfat dry milk in Tris-buffered saline: 10 mM Tris (pH 7.5), 100 mM NaCl) for 1 h. After blocking, membranes were incubated with monoclonal antihuman Dkk-1 antibodies (R&D Systems) or IGFBP-1 antibodies (Santa Cruz Biotechnology) at a 1:200 dilution in blocking buffer overnight at 4 C. After 3 x 15 min washes in Tris-buffered saline buffer, membranes were incubated with the goat-antimouse secondary antibody (Upstate, Lake Placid, NY) at 1:1000 dilution for 1 h. Bound antibodies were detected using the ECL-Plus chemiluminescent detection system (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) and exposed to x-ray films (Eastman Kodak).

Statistical analysis

Displayed data are expressed as means ± SEM. One-way ANOVA, followed by Tukey’s test as post hoc was used to evaluate the effects of treatments, after exposure for 3, 24, 48, and 72 h, on Dkk-1 mRNA expression vs. control. Effects of E2P4, RU486 addition to the E2P4 treatment (RU486+ E2P4), and E2P4 withdrawal from the media (no E2P4) on Dkk-1 mRNA expression in human endometrial stromal cells were analyzed by one-way ANOVA followed by Tukey’s test as post hoc. In each analysis, differences were considered significant if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Short-term cultures of human endometrial stromal cells

Regulation of Dkk-1 mRNA and protein expression by progesterone or 8-bromo cAMP in human endometrial stromal cells. When human endometrial stromal cells were treated with E2P4 for 24, 48, or 72 h, up-regulation of Dkk-1 mRNA expression was observed after 24 h, increasing 5- to 6-fold over the time of treatment, as determined by dot-blot analysis (Fig. 1Go, A and B). E2 alone or treatment with 8-bromo cAMP for 24, 48, or 72 h revealed no regulation of Dkk-1 mRNA expression. Further confirmation of mRNA regulation by progesterone was validated by quantitative RT-PCR (Fig. 1CGo). Stromal cells treated with E2P4 for 3, 24, 48, and 72 h showed a significant increase (approximately 7-fold by 72 h of treatment) in Dkk-1 mRNA expression. E2 treatment did not have an effect on Dkk-1 mRNA expression (Fig. 1CGo).


Figure 1
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  		FIG. 1. Regulation of Dkk-1 mRNA expression in short-term endometrial stromal cells cultures. A, Representative Dkk-1 mRNA expression obtained by dot-blot analysis in endometrial stromal cells treated with E2, E2P4, or 8-bromo cAMP for 3, 24, 48, and 72 h and compared with the nontreated control groups (–). B, Densitometric analysis showed no regulation of Dkk-1 mRNA by E2 or 8-bromo cAMP over 24, 48, or 72 h. Treatment with E2P4 induced up-regulation of Dkk-1 mRNA expression. All data were normalized to GAPDH as a constitutively expressed marker and are presented as a percentage of GAPDH ± SEM of four different dot-blot experiments. C, Dkk-1 regulation in endometrial stromal cells determined by quantitative RT-PCR after 3, 24, 48, and 72 h of treatment with 10 nM E2 or 1 µM progesterone and 10 nM E2 (E2P4) in serum-free medium and compared with the no-treatment controls. Data represent mean relative expression of Dkk-1 in endometrial stromal cells isolated from three different subjects ± SEM. *, P < 0.05.

 
Regulation of Dkk-1 protein was investigated in media conditioned by endometrial stromal cells under the same conditions in which the mRNA expression studies were conducted. Figure 2AGo illustrates that significant secretion of Dkk-1 protein into the media was observed after 72 h of treatment with E2P4, and minimal secretion of Dkk-1 was detected in the control, E2, and 8-bromo cAMP treatment groups. RU486 effectively blocks the secretion of Dkk-1 into the CM after 72 h of treatment (Fig. 2BGo). The secreted Dkk-1 appeared as five bands in the Western blot analysis. The three bands of higher apparent molecular weight represent different glycosylated forms, as demonstrated by N-glycosidase F treatment of the CM (Fig. 3Go). One band corresponds to the nonglycosylated Dkk-1 (at approximately 27 kDa), and a minor fraction of Dkk-1 appeared to be partially degraded, as evidenced by a band of a lower apparent molecular weight (Figs. 2Go, A and B, and 3Go). We could not detect any Dkk-1 protein in the cell lysates from human endometrial stromal cells cultured in serum-free media, E2, or 8-bromo cAMP-treated cells after 72 h of treatment (data not shown).


Figure 2
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  		FIG. 2. Regulation of Dkk-1 protein secretion in short-term endometrial stromal cell cultures. A, Western blot analysis of secreted Dkk-1 protein in CM obtained after 72 h of no treatment (–) and CM obtained from human endometrial stromal cells treated with E2, 1 µM progesterone and 10 nM E2 (E2P4), or 1 mM 8-bromo cAMP. Arrows indicate glycosylated forms of the protein (three upper arrows), nonglycosylated form (at approximately 27 kDa), and the lower band (at ~17 kDa) representing partial degradation of Dkk-1 protein. B, Western blot analysis of secreted Dkk-1 protein in CM obtained after 72 h of no additives (–) and CM obtained from endometrial stromal cells treated with RU486, E2P4, and E2P4+RU486. Figures are representative of three different experiments.

 

Figure 3
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  		FIG. 3. Enzymatic deglycosylation of secreted Dkk-1 protein in CM obtained from human endometrial stromal cells. Lane a: Dkk-1 protein secreted into the CM appeared as five bands in the Western blot analysis, three of higher apparent molecular weight, representing different glycosylated forms of the protein, one band corresponding to the nonglycosylated Dkk-1 (at 27 kDa), and the bottom band representing partial degradation of the protein. Lane b: In CM secreted Dkk-1 protein treated with N-glycosidase F revealed one band as seen in the cell lysates (~27 kDa) (compare lanes b and c). Lane c: Dkk-1 protein in cell lysates (from stromal cells that were treated with E2P4 for 17 d) migrates at approximately 27 kDa. Lane d: Glycosylated recombinant human Dkk-1 protein (100 ng/lane) migrates at approximately 37 kDa.

 
Long-term cultures of human endometrial stromal cells

Progesterone regulation of Dkk-1. When stromal cells were treated with E2P4 for up to 14 d and total RNA isolated on d 2, 4, 6, 10, and 14 of treatment, up-regulation of Dkk-1 mRNA expression was observed by Northern blot analysis, beginning at 2 d of treatment and increasing through 14 d of treatment (Fig. 4AGo). Real-time RT-PCR data obtained from RNA isolated from human endometrial stromal cells from five subjects, after 17 d of treatment with E2P4, showed significant (33-fold) up-regulation of Dkk-1 mRNA expression, compared with the nondecidualized control group. Furthermore, no regulation of Dkk-1 mRNA expression was observed when cells were treated with 8-bromo cAMP for 3 d (Fig. 4BGo). Endometrial stromal cells treated with 8-bromo cAMP for 3 d or progesterone for 17 d produced about the same amount of IGFBP-1 (>1 µg/ml) in the CM.


Figure 4
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  		FIG. 4. Regulation of Dkk-1 mRNA expression in long-term endometrial stromal cultures. A, Dkk-1 mRNA expression in human endometrial stromal cells after 2, 4, 6, 10, and 14 d treatment with E2P4, compared with the no-treatment control (–) and determined by Northern blot analysis. Total RNA (20 µg) was isolated from endometrial stromal cells in control (no-treatment group) and after treatment with E2P4 and hybridized with radiolabeled Dkk-1 cDNA probe. For the loading control, the blot was rehybridized with radiolabeled GAPDH cDNA probe (see Subjects and Methods). B, Expression of Dkk-1 mRNA obtained by quantitative RT-PCR from cultured endometrial stromal cells treated with E2P4 for 17 d. Fold regulation in treated samples was obtained by comparison with nontreated controls (endometrial stromal cells grown in serum-free media for the equivalent amount of time) after normalization with 18S (as a constitutively expressed marker). Comparison is made with 8-bromo cAMP treatment of endometrial stromal cells for 72 h, which had equivalent production of the decidual marker, IGFBP-1 (see Subjects and Methods). Data represent mean of the relative expression of Dkk-1 mRNA in endometrial stromal cells isolated from five different subjects ± SEM. ***, P < 0.001.

 
Effects of RU486 and progesterone withdrawal on Dkk-1 mRNA and protein expression in human endometrial stromal cells

At d 14 of E2P4 treatment (t = 0), endometrial stromal cells were decidualized (confirmed by levels of IGFBP-1 in the CM being > 1 µg/ml), and further treatment with E2P4, for an additional 3, 6, and 9 d resulted (Fig. 5AGo) in significant Dkk-1 mRNA up-regulation, compared with the nondecidualized control group, and determined by quantitative RT-PCR. When RU486 was added to the E2P4 treatment of decidualized stromal cells (i.e. after 14 d of E2P4 treatment or t = 0), there was a significant inhibition of Dkk-1 mRNA expression observed after 6 and 9 d of treatment (Fig. 5AGo). Similarly, in the group in which E2P4 was withdrawn from the media, Dkk-1 mRNA was found to be significantly down-regulated to the control levels of nondecidualized cells in serum-free medium (Fig. 5AGo). These changes in Dkk-1 mRNA expression paralleled changes of IGFBP-1 protein secreted into the CM, again demonstrating inhibition of progesterone action by RU486 or the effects of E2P4 withdrawal (Fig. 5BGo).


Figure 5
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  		FIG. 5. Effects of progesterone, RU486, and E2P4 withdrawal on Dkk-1 and the decidual marker IGFBP-1 expression. A, Effects of E2P4, RU486 addition to the E2P4 treatment (RU486+E2P4), and E2P4 withdrawal from the media (no E2P4) on Dkk-1 mRNA expression in human endometrial stromal cells determined by quantitative RT-PCR. Data were normalized to 18S as an internal control, and expression was compared with the nontreated controls (endometrial stromal cells in serum-free media with addition of vehicle control for the equivalent time). Data are presented as a fold change in Dkk-1 mRNA expression in five different subjects ± SEM and compared with the E2P4 treatment for the statistical significance. ***, P < 0.001. B, IGFBP-1 protein levels in the CM after treatment of stromal cells with E2P4, addition of RU486 to E2P4 treatment (RU486+E2P4), or E2P4 withdrawal (no E2P4) at 3, 6, and 9 d after decidualization. Data are shown as a nanograms per milliliter of IGFBP-1 protein expression (determined by ELISA) in CM from five different subjects ± SEM.

 
Western blot analysis of Dkk-1 protein levels in endometrial stromal cell lysates and CM (Fig. 6Go) confirmed the regulation observed at the mRNA level. Dkk-1 protein production was maintained when cells were continuously treated with E2P4, whereas when RU486 was added to E2P4 treatment or E2P4 was withdrawn from the media, a significant decrease of Dkk-1 protein levels was evident in the endometrial stromal cells (Fig. 6AGo) as well as the CM (Fig. 6BGo). These changes paralleled changes in IGFBP-1 secretion into CM under similar conditions (Fig. 6CGo).


Figure 6
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  		FIG. 6. Dkk-1 protein regulation in long-term cultures of endometrial stromal cells. Dkk-1 protein expression in human endometrial stromal cells treated with E2P4 for 14 d (t = 0) and lysed at 3, 6, and 9 d after decidualization; addition of RU486 to E2P4 treatment (RU486+E2P4), or E2P4 withdrawal (no E2P4) was determined by Western blotting. A, Cell lysates. Dkk-1 protein levels were high in lysates obtained from the cells treated with E2P4. A significant decrease of Dkk-1 protein production was observed with addition of RU486 to the E2P4 treatment or when E2P4 was withdrawn from the media. B, CM. High levels of Dkk-1 protein in the media were maintained when endometrial stromal cells were continuously treated with E2P4; whereas a significant decrease of secreted Dkk-1 protein is observed with addition of RU486 to the E2P4-treated group or when E2P4 was withdrawn from the media. C, IGFBP-1. IGFBP-1 protein levels in the CM obtained from the endometrial stromal cells treated with E2P4, addition of RU486 to E2P4 treatment, and E2P4 withdrawal after 3, 6, and 9 d after cells have been decidualized, parallel Dkk-1 levels in the cell lysates as well as in CM. All figures are representative of three different experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Progesterone is an essential hormone for reproductive functions, including ovulation, implantation, and pregnancy. It acts through its nuclear receptors (progesterone receptor) derived from the same gene (42, 43), although it may also have membrane receptor-mediated effects (44). Progesterone acts in human endometrium directly on endometrial stromal cells, regulating gene expression (45, 46), and indirectly on epithelial cells by increasing the level of growth factors secreted by the stromal cells that subsequently regulate epithelial gene expression in a paracrine manner. Marked changes in expression levels of numerous genes in human endometrium during the window of implantation (peak circulating progesterone levels), compared with times of the cycle with low progesterone (14, 16), or high E2 (13, 16) suggest hormonal control of these genes. We previously demonstrated that the expression of two of the major inhibitors of the Wnt signaling pathway, Dkk-1 and frpHE, is cycle dependent in human endometrium (18). frpHE is highly up-regulated in proliferative, compared with secretory, endometrium and is markedly up-regulated in E2-dependent endometrial and breast carcinomas (13, 18, 47, 48). Dkk-1, on the other hand, is up-regulated in the midsecretory phase, compared with the proliferative phase, suggesting that progesterone may regulate its gene expression in normal endometrium.

In the current investigation, an in vitro endometrial stromal cell culture system was used to study possible short-term and long-term progesterone regulation of Dkk-1. The present study demonstrates that progesterone, but not cAMP (or E2), regulates Dkk-1 mRNA and protein levels in cultured endometrial stromal cells. We observed up-regulation of Dkk-1 mRNA within 3–72 h of E2P4 treatment, and after 17 d of E2P4 treatment, there was 33-fold up-regulation of Dkk-1 mRNA. Significant up-regulation of Dkk-1 by E2P4 treatment was observed also at the protein level. E2P4 withdrawal from the media, or addition of RU486 to E2P4 treatment, resulted in a decrease of Dkk-1 mRNA and protein to basal levels. Inhibition of progesterone regulation of Dkk-1 mRNA and protein by RU486 strongly supports regulation of Dkk-1 expression in human endometrium involving the progesterone nuclear receptor. It is unclear, however, whether the progesterone-mediated regulation of Dkk-1 expression in endometrial stromal cells at the transcriptional and translational levels is direct, or if it is indirect, acting through unknown mediator(s). The rapid response to progesterone (i.e. within 3 h, in contrast to classical markers of progesterone action on stromal cells that are usually regulated after 8–10 d of treatment) and the lack of cAMP regulation (see below) further suggest that progesterone acts directly through the progesterone receptor for the regulation of Dkk-1 in this cell type. Analysis of the Dkk-1 promoter region does not reveal progesterone response element consensus sequences, which would support that progesterone may act through secondary routes or induction of unknown transcription factors to enhance Dkk-1 production. It has been shown that some decidua-specific genes (including prolactin) are regulated by progesterone directly (or at the transcriptional level) without having consensus sequences for progesterone response elements in their promoters (36, 37, 49).

Interestingly, Dkk-1 mRNA and protein levels were not regulated by treatment of endometrial stromal cells with 8-bromo cAMP, another known inducer of the decidual phenotype. Endometrial stromal cells treated with 8-bromo cAMP for 3 d or progesterone for 17 d produced about the same amount of IGFBP-1 in the CM, demonstrating that the decidualization process of the two treatment groups was approximately the same, with this marker as an end point. It has been shown that progestin treatment of primary endometrial stromal cultures does elicit modest expression of decidual markers (IGFBP-1 and prolactin), but only after several days of treatment by which time the intracellular levels of cAMP are increasing (41). However, the finding that progesterone up-regulates Dkk-1 expression whereas 8-bromo cAMP does not, despite equivalent decidualization marker expression under those treatments, suggests that Dkk-1 is not regulated through the protein kinase A pathway in human endometrium. Studies by others (12, 37, 46) have shown some genes in human endometrial stromal cells that are uniquely up-regulated by progesterone but not cAMP. This raises an interesting possibility of diversion of the cAMP and progesterone regulatory pathways during decidualization of human endometrial stromal cells.

The importance of Wnt signaling has been demonstrated in the development of functional endometrium in a mouse model. Wnt-7a null (–/–) mice do not form endometrial glands and are infertile (50), and Wnt-4 is crucial for sex-specific development of the female reproductive tract (51). Wnt-5a is expressed in the uterine mesenchyme and is required for the generation of the uterine glands for cellular and molecular responses to the exogenous E2 and regulation of Wnt-7a and Hox genes (52, 53). These studies underscore the importance of Wnt signaling in mediating mesenchymal-epithelial interactions in the murine endometrium, and specific roles for the Wnt pathway in human endometrium are under investigation in our laboratory.

The physiological relevance of Dkk-1 up-regulation in response to progesterone is not fully understood. It is known, in mice, that the Wnt family of proteins, including their receptors and antagonists, contribute to attachment of the blastocyst to uterine luminal epithelium and synthesis of extracellular matrix components associated with decidualization (54). Mouse blastocysts express genes encoding several members of the Wnt signaling pathway (Wnt-5a and Wnt-11), underscoring Wnts as potential mediators of embryo-uterine communication during implantation (55). These interactions primarily involve blastocyst-maternal endometrial epithelial interactions. We have shown previously, by in situ hybridization, that Dkk-1 is expressed in human endometrial stromal cells (18), and in this study we have demonstrated how Dkk-1 is regulated by progesterone, but not cAMP, in cultured human endometrial stromal cells. We speculate that stromal Dkk-1 may affect paracrine and, also, autocrine Wnt signaling by inhibiting the Wnt pathway and therefore regulating signaling at a certain time of the menstrual cycle or have a Wnt-independent function. In normal human endometrium in vivo, Dkk-1 mRNA regulation is dependent on progesterone, and its expression increases in the early-secretory phase, peaking in the midsecretory and decreasing in late-secretory phase (shown by microarray analysis and quantitative PCR) (56). This additionally confirms the regulation of Dkk-1 by progesterone and its possible role during the window of implantation.

The cumulative data from this study and others lead us to propose that there is a balance among factors secreted by the endometrium and the blastocyst during the early stages of implantation that influence blastocyst attachment, development, and invasion and that the Wnt signaling pathway plays a role, yet to be fully understood, in these processes. Dkk-1 likely functions to limit Wnt actions during the establishment of endometrial receptivity to implantation and in the early stages of embryonic implantation and trophoblast invasion into the stroma.


   Footnotes
 
This work was supported by a fellowship grant from Diagnostic Systems Laboratories and The Alfred Benzon Foundation (to M.T.O.), and the National Institutes of Health Specialized Cooperative Centers Program in Reproduction Research (National Institute of Child Health and Human Development 31398-09) (to L.C.G.).

S.T., M.T.O., A.E.H., N.L.J., E.S., and L.C.G. have nothing to declare.

First Published Online January 31, 2006

Abbreviations: CM, Conditioned media; Dkk-1, Dickkopf-1; E2, estradiol; E2P4, progesterone + E2; frpHE, frizzled related protein HE; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IGFBP, IGF binding protein.

Received April 8, 2005.

Accepted January 25, 2006.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Hess AP, Nayak NR, Giudice LC 2006 Oviduct and endometrium: cyclic changes in the primate oviduct and endometrium. In: Knobil E, Neill JD, eds. The physiology of reproduction, 3rd ed. San Diego: Academic Press; 359–403
  2. Tabanelli S, Tang B, Gurpide E 1992 In vitro decidualization of human endometrial stromal cells. J Steroid Biochem Mol Biol 42:337–344[CrossRef][Medline]
  3. Tang B, Guller S, Gurpide E 1994 Mechanism of human endometrial stromal cells decidualization. Ann NY Acad Sci 734:19–25[Medline]
  4. Chen G, Huang JR, Mazella J, Tseng L 1989 Long-term effects of progestin and RU 486 on prolactin production and synthesis in human endometrial stromal cells. Hum Reprod 4:355–358[Abstract/Free Full Text]
  5. Huang JR, Tseng L, Bischof P, Janne OA 1987 Regulation of prolactin production by progestin, estrogen, and relaxin in human endometrial stromal cells. Endocrinology 121:2011–2017[Abstract]
  6. Bell SC, Jackson JA, Ashmore J, Zhu HH, Tseng L 1991 Regulation of insulin-like growth factor-binding protein-1 synthesis and secretion by progestin and relaxin in long term cultures of human endometrial stromal cells. J Clin Endocrinol Metab 72:1014–1024[Abstract]
  7. Giudice LC, Milkowski DA, Lamson G, Rosenfeld RG, Irwin JC 1991 Insulin-like growth factor binding proteins in human endometrium: steroid-dependent messenger ribonucleic acid expression and protein synthesis. J Clin Endocrinol Metab 72:779–787[Abstract]
  8. Irwin JC, Kirk D, King RJ, Quigley MM, Gwatkin RB 1989 Hormonal regulation of human endometrial stromal cells in culture: an in vitro model for decidualization. Fertil Steril 52:761–768[Medline]
  9. Zhu HH, Huang JR, Mazela J, Elias J, Tseng L 1992 Progestin stimulates the biosynthesis of fibronectin and accumulation of fibronectin mRNA in human endometrial stromal cells. Hum Reprod 7:141–146[Abstract/Free Full Text]
  10. Kim JJ, Jaffe RC, Fazleabas AT 1998 Comparative studies on the in vitro decidualization process in the baboon (Papio anubis) and human. Biol Reprod 59:160–168[Abstract/Free Full Text]
  11. Telgmann R, Maronde E, Tasken K, Gellersen B 1997 Activated protein kinase A is required for differentiation-dependent transcription of the decidual prolactin gene in human endometrial stromal cells. Endocrinology 138:929–937[Abstract/Free Full Text]
  12. Mizuno K, Tanaka T, Umesaki N, Ogita S 1998 Establishment and characterization of in vitro decidualization in normal human endometrial stromal cells. Osaka City Med J 44:105–115[Medline]
  13. Kao LC, Tulac S, Lobo S, Imani B, Yang JP, Germeyer A, Osteen K, Taylor RN, Lessey BA, Giudice LC 2002 Global gene profiling in human endometrium during the window of implantation. Endocrinology 143:2119–2138[Abstract/Free Full Text]
  14. Riesewijk A, Martin J, van Os R, Horcajadas JA, Polman J, Pellicer A, Mosselman S, Simon C 2003 Gene expression profiling of human endometrial receptivity on days LH+2 versus LH+7 by microarray technology. Mol Hum Reprod 9:253–264[Abstract/Free Full Text]
  15. Horcajadas JA, Riesewijk A, Martin J, Cervero A, Mosselman S, Pellicer A, Simon C 2004 Global gene expression profiling of human endometrial receptivity. J Reprod Immunol 63:41–49[CrossRef][Medline]
  16. Carson DD, Lagow E, Thathiah A, Al-Shami R, Farach-Carson MC, Vernon M, Yuan L, Fritz MA, Lessey B 2002 Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening. Mol Hum Reprod 8:871–879[Abstract/Free Full Text]
  17. Borthwick JM, Charnock-Jones DS, Tom BD, Hull ML, Teirney R, Phillips SC, Smith SK 2003 Determination of the transcript profile of human endometrium. Mol Hum Reprod 9:19–33[Abstract/Free Full Text]
  18. Tulac S, Nayak NR, Kao LC, Van Waes M, Huang J, Lobo S, Germeyer A, Lessey BA, Taylor RN, Suchanek E, Giudice LC 2003 Identification, characterization, and regulation of the canonical Wnt signaling pathway in human endometrium. J Clin Endocrinol Metab 88:3860–3866[Abstract/Free Full Text]
  19. Wodarz A, Nusse R 1998 Mechanisms of Wnt signaling in development. Annu Rev Cell Dev Biol 14:59–88[CrossRef][Medline]
  20. Nusse R, Varmus HE 1992Wnt genes. Cell 69:1073–1087
  21. Parr BA, McMahon AP 1994 Wnt genes and vertebrate development. Curr Opin Genet Dev 4:523–528[CrossRef][Medline]
  22. Crawford HC, Fingleton BM, Rudolph-Owen LA, Goss KJ, Rubinfeld B, Polakis P, Matrisian LM 1999 The metalloproteinase matrilysin is a target of ß-catenin transactivation in intestinal tumors. Oncogene 18:2883–2891[CrossRef][Medline]
  23. He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT, Morin PJ, Vogelstein B, Kinzler KW 1998 Identification of c-MYC as a target of the APC pathway. Science 281:1509–1512[Abstract/Free Full Text]
  24. Mann B, Gelos M, Siedow A, Hanski ML, Gratchev A, Ilyas M, Bodmer WF, Moyer MP, Riecken EO, Buhr HJ, Hanski C 1999 Target genes of ß-catenin-T cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas. Proc Natl Acad Sci USA 96:1603–1608[Abstract/Free Full Text]
  25. Willert J, Epping M, Pollack JR, Brown PO, Nusse R 2002 A transcriptional response to Wnt protein in human embryonic carcinoma cells. BMC Dev Biol 2:8
  26. Shtutman M, Zhurinsky J, Simcha I, Albanese C, D’Amico M, Pestell R, Ben-Ze’ev A 1999 The cyclin D1 gene is a target of the ß-catenin/LEF-1 pathway. Proc Natl Acad Sci USA 96:5522–5527[Abstract/Free Full Text]
  27. Fujita M, Ogawa S, Fukuoka H, Tsukui T, Nemoto N, Tsutsumi O, Ouchi Y, Inoue S 2002 Differential expression of secreted frizzled-related protein 4 in decidual cells during pregnancy. J Mol Endocrinol 28:213–223[Abstract]
  28. Bafico A, Gazit A, Pramila T, Finch PW, Yaniv A, Aaronson SA 1999 Interaction of frizzled related protein (FRP) with Wnt ligands and the frizzled receptor suggests alternative mechanisms for FRP inhibition of Wnt signaling. J Biol Chem 274:16180–16187[Abstract/Free Full Text]
  29. Shou J, Ali-Osman F, Multani AS, Pathak S, Fedi P, Srivenugopal KS 2002 Human Dkk-1, a gene encoding a Wnt antagonist, responds to DNA damage and its overexpression sensitizes brain tumor cells to apoptosis following alkylation damage of DNA. Oncogene 21:878–889[CrossRef][Medline]
  30. Fedi P, Bafico A, Nieto Soria A, Burgess WH, Miki T, Bottaro DP, Kraus MH, Aaronson SA 1999 Isolation and biochemical characterization of the human Dkk-1 homologue, a novel inhibitor of mammalian Wnt signaling. J Biol Chem 274:19465–19472[Abstract/Free Full Text]
  31. Bafico A, Liu G, Yaniv A, Gazit A, Aaronson SA 2001 Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/arrow. Nat Cell Biol 3:683–686[CrossRef][Medline]
  32. Glinka A, Wu W, Delius H, Monaghan AP, Blumenstock C, Niehrs C 1998 Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature 391:357–362[CrossRef][Medline]
  33. Monaghan AP, Kioschis P, Wu W, Zuniga A, Bock D, Poustka A, Delius H, Niehrs C 1999 Dickkopf genes are co-ordinately expressed in mesodermal lineages. Mech Dev 87:45–56[CrossRef][Medline]
  34. Grotewold L, Theil T, Ruther U 1999 Expression pattern of Dkk-1 during mouse limb development. Mech Dev 89:151–153[CrossRef][Medline]
  35. Wang J, Shou J, Chen X 2000 Dickkopf-1, an inhibitor of the Wnt signaling pathway, is induced by p53. Oncogene 19:1843–1848[CrossRef][Medline]
  36. 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]
  37. Gellersen B, Brosens J 2003 Cyclic AMP and progesterone receptor cross-talk in human endometrium: a decidualizing affair. J Endocrinol 178:357–372[Abstract]
  38. Kirk D, Irwin JC 1980 Normal human endometrium in cell culture. Methods Cell Biol 21B:51–77
  39. Irwin JC, Utian WH, Eckert RL 1991 Sex steroids and growth factors differentially regulate the growth and differentiation of cultured human endometrial stromal cells. Endocrinology 129:2385–2392[Abstract]
  40. Tang B, Guller S, Gurpide E 1993 Mechanisms involved in the decidualization of human endometrial stromal cells. Acta Eur Fertil 24:221–223[Medline]
  41. Brar AK, Frank GR, Kessler CA, Cedars MI, Handwerger S 1997 Progesterone-dependent decidualization of the human endometrium is mediated by cAMP. Endocrine 6:301–307[Medline]
  42. Mote PA, Balleine RL, McGowan EM, Clarke CL 1999 Colocalization of progesterone receptors A and B by dual immunofluorescent histochemistry in human endometrium during the menstrual cycle. J Clin Endocrinol Metab 84:2963–2971[Abstract/Free Full Text]
  43. Mote PA, Balleine RL, McGowan EM, Clarke CL 2000 Heterogeneity of progesterone receptors A and B expression in human endometrial glands and stroma. Hum Reprod 15(Suppl 3):48–56
  44. Zhu Y, Rice CD, Pang Y, Pace M, Thomas P 2003 Cloning, expression, and characterization of a membrane progestin receptor and evidence it is an intermediary in meiotic maturation of fish oocytes. Proc Natl Acad Sci USA 100:2231–2236[Abstract/Free Full Text]
  45. 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]
  46. Popovici RM, Kao LC, Giudice LC 2000 Discovery of new inducible genes in in vitro decidualized human endometrial stromal cells using microarray technology. Endocrinology 141:3510–3513[Abstract/Free Full Text]
  47. Abu-Jawdeh G, Comella N, Tomita Y, Brown LF, Tognazzi K, Sokol SY, Kocher O 1999 Differential expression of frpHE: a novel human stromal protein of the secreted frizzled gene family, during the endometrial cycle and malignancy. Lab Invest 79:439–447[Medline]
  48. Hrzenjak A, Tippl M, Kremser ML, Strohmeier B, Guelly C, Neumeister D, Lax S, Moinfar F, Tabrizi AD, Isadi-Moud N, Zatloukal K, Denk H 2004 Inverse correlation of secreted frizzled-related protein 4 and ß-catenin expression in endometrial stromal sarcomas. J Pathol 204:19–27[CrossRef][Medline]
  49. Lessey BA 2003 Two pathways of progesterone action in the human endometrium: implications for implantation and contraception. Steroids 68:809–815[CrossRef][Medline]
  50. Miller C, Sassoon DA 1998 Wnt-7a maintains appropriate uterine patterning during the development of the mouse female reproductive tract. Development 125:3201–3211[Abstract]
  51. Vainio S, Heikkila M, Kispert A, Chin N, McMahon AP 1999 Female development in mammals is regulated by Wnt-4 signalling. Nature 397:405–409[CrossRef][Medline]
  52. Mericskay M, Kitajewski J, Sassoon D 2004 Wnt5a is required for proper epithelial-mesenchymal interactions in the uterus. Development 131:2061–2072[Abstract/Free Full Text]
  53. Miller C, Pavlova A, Sassoon DA 1998 Differential expression patterns of Wnt genes in the murine female reproductive tract during development and the estrous cycle. Mech Dev 76:91–99[CrossRef][Medline]
  54. Kierszenbaum AL 2001 Decidualization and implantation: embryo-uterine bioinformatics at work. Mol Reprod Dev 59:123–125[CrossRef][Medline]
  55. Mohamed OA, Dufort D, Clarke HJ 2004 Expression and estradiol regulation of Wnt genes in the mouse blastocyst identify a candidate pathway for embryo-maternal signaling at implantation. Biol Reprod 71:417–424[Abstract/Free Full Text]
  56. Talbi S, Hamilton AE, Vo KC, Tulac S, Overgaard MT, Dosiou C, Shay NL, Nezhat CN, Kempson R, Lessey BA, Nayak NR, Giudice LC 2006 Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women. Endocrinology 147:1097–1121[Abstract/Free Full Text]



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