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Department of Gynecology and Obstetrics (S.T., N.R.N., L.C.K., J.H., S.L., A.G., L.C.G.), Stanford University, Stanford, California 94305-5317; Stratagene (M.v.W.), La Jolla, California 92037; Department of Obstetrics & Gynecology (B.A.L.), University of North Carolina, Chapel Hill, North Carolina 27599; Department of Obstetrics, Gynecology, and Reproductive Sciences (R.N.T.), University of California, San Francisco, California 94143; and Department of Obstetrics and Gynecology (E.S.), Zagreb University School of Medicine, 10000 Zagreb, Croatia
Address all correspondence and requests for reprints to: Linda C. Giudice, M.D., Ph.D., Center for Research on Women’s Health and Reproductive Medicine, Division of Reproductive Endocrinology and Infertility, Department of Gynecology and Obstetrics, Stanford University, Stanford, California 94305-5317. E-mail: giudice{at}stanford.edu.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Secreted Wnt proteins comprise a family of highly conserved glycoproteins that exhibit pivotal roles in cell proliferation and differentiation, epithelial-mesenchymal communication, and embryogenesis (5, 6, 7). They bind and act through cell surface receptors known as Frizzled (Fz), a large family of seven membrane-spanning domain receptors (8). Wnt/Fz interactions at the cell surface activate Dishevelled (Dvl) leading to the inactivation of glycogen synthase kinase-3ß (GSK-3ß) by phosphorylation. When activated, GSK-3ß phosphorylates adenomatous polyposis coli and Axin, increasing their binding activities for ß-catenin, marking it for degradation by N-terminal phosphorylation (9, 10, 11). GSK-3ß inactivation, in response to Wnt, dissociates this complex formation subsequently leading to cytoplasmic ß-catenin accumulation and entry into the nucleus where it regulates gene transcription (12, 13, 14). Recent studies have identified several families of inhibitors of Wnt signaling. Secreted frizzled-related proteins share structural similarities to the Frizzled receptor family of proteins and antagonize Wnt actions at the level of receptor-ligand binding (15, 16). Another inhibitor, Dickkopf (Dkk), binds to low-density lipoprotein receptor-related protein-6 (LRP-6), a coreceptor for Wnt, and inhibits the Wnt signaling pathway. Recent work suggests that Dkk interacts with the coreceptors LRP5/6 and inhibits signaling by disrupting the binding of LRP6 to the Wnt/Fz ligand-receptor complex (17, 18). Some of the target genes for Wnt signaling, including Hoxa-10, Hoxa-11, cyclooxygenase-2, and peroxisome proliferator activated receptor-
, (http://www.stanford.edu/
rnusse/pathways/targets.html) are known to be important in decidualization of the endometrium and in implantation in the mouse (19, 20, 21).
Wnt genes are responsive to circulating sex steroids in several steroid-responsive organs including the female reproductive tract. In adult mouse, Wnt-7a is exclusively expressed in the uterine luminal, but not glandular, epithelium and acts to regulate the boundaries of expression of other Wnt ligands to establish the correct developmental axis of the uterus (22). Wnt-7a-/- mice are infertile and have complete absence of uterine glands. In human endometrium, Wnt-2, Wnt-3, Wnt-4, Wnt-5a, Wnt-7a, and Wnt-7b are constitutively expressed throughout the menstrual cycle, and down-regulation of Wnt-2, Wnt-3, Wnt-4, and Wnt-5a has been observed in endometrial carcinoma (23). These observations, in addition to several microarray studies including a previous report from our laboratory (2, 3, 4), indicate regulation of Wnt family genes in human endometrium during the menstrual cycle. Herein, we examine the expression of several Wnt ligands, receptors, inhibitors, and downstream effectors in human proliferative and secretory phase endometrium. Our results on the cell-specific expression and cycle-dependent regulation of select members of the Wnt family in human endometrium suggest important roles in mediating mesenchymal-epithelial interactions during endometrial differentiation and possibly implantation.
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Materials and Methods |
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Endometrial biopsies were obtained from normally cycling women after informed consent, under an approved protocol by the Stanford University Committee on the Use of Human Subjects in Medical Research, and the Human Subjects Committees at the University of North Carolina and the University of California, San Francisco. All specimens were obtained in accordance with the Declaration of Helsinki. A total of 12 biopsy samples was obtained from two time points of the menstrual cycle: six in the mid- to late proliferative phase (peak circulating estradiol levels) and six in mid-secretory phase (peak estradiol and progesterone). Due to limitation in tissue availability, six of the samples (three in the mid- to late proliferative and three in the mid-secretory phase endometrium) were used for RT-PCR and real-time PCR studies, whereas six additional samples were used for in situ hybridization. All subjects in the age group between 28 and 39 yr had regular menstrual cycles (26–35 d), were documented not to be pregnant, had no history of endometriosis, and had not been on hormonal treatment for at least 3 months before biopsy.
RNA extraction and reverse transcription (RT)
Total RNA from endometrial tissue (in the proliferative and secretory phases of the cycle) was isolated using Trizol (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. Isolated total RNA was then treated with deoxyribonuclease and purified by RNeasy Spin Columns (QIAGEN, Valencia, CA). RNA integrity was verified by agarose gel electrophoresis/ethidium bromide staining and by OD260/280 absorption ratio greater than 1.95. Total RNA (1 µg) was reverse transcribed using Omniscript kit (QIAGEN) according to the manufacturer’s instructions with a 1:1 ratio of oligo (deoxythymidine)16–18 and random hexamers (Invitrogen).
PCR
RT products were used in PCRs containing MgCl2 (2.5 mM), deoxynucleotide triphosphates (0.1 mM), 1x PCR buffer, Taq polymerase, or HotStarTaq (QIAGEN) (2.5 U), and specific primer pairs (30 pmol) using the Eppendorf Mastercycler Gradient (Eppendorf, Hamburg, Germany). Primer sequences (Table 1
) were designed from public databases and synthesized at the Stanford University School of Medicine Protein and Nucleic Acid (PAN) Facility. Wnt-7a, FrpHE, Fz-6, Dkk-1, and ß-catenin PCR products were subcloned into pDrive Cloning Vectors (QIAGEN) by TA cloning (using primer pairs from Table 1) to generate specific probes for in situ hybridization. The identity of each PCR product was confirmed by sequencing at the Stanford PAN facility.
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Primers for target and reference genes were designed using the PCR design software at http://labtools.stratagene.com and synthesized by QIAGEN (Table 2). Real-time PCRs were performed in quadruplicates using the QuantiTect SYBR Green PCR Kit (QIAGEN) following the manufacturer’s instructions and carried out in the Mx4000 Q-PCR system (Stratagene, La Jolla, CA). All assays were optimized for primer concentration and PCR product specificity based on melting curve analysis and gel electrophoresis. Each real-time PCR was comprised of 250 ng of template cDNA and optimized primers at the concentrations of 50–250 nM. The thermal cycling conditions included an initial activation step at 95 C for 10 min, followed by 40 cycles of denaturation, annealing and amplification (95 C for 30 sec, 60 C for 1 min, 72 C for 30 sec). PCR products were analyzed by thermal dissociation (55–95 C) with a fluorescence measurement at every 1-degree increment. For each assay, a no-template and no-RT controls were included to verify the quality and cDNA specificity of the primers. Reactions were verified by agarose gel electrophoresis of PCR products and showed single correctly sized bands for each sample or no products for the negative controls. The PCR amplification efficiency of the reactions and correlation coefficients were determined from serially diluted human endometrial cDNA (250, 62.5, 15.6, and 3.9 ng), using the slope of the best-fit curve for Ct vs. concentration, and were calculated by the Mx4000 software. Investigated transcripts showed high efficiency of amplification and a linear response. No primer-dimer formation was observed during the 40 PCR amplification cycles. No significant difference was observed in the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) between proliferative and secretory phases using the same starting amount of template, indicating that this is an appropriate reference gene.
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Ct) using the following mathematical model (25):
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CtGOI and CtRef are the differences in threshold values between proliferative and secretory phase samples for the gene of interest and the normalizer. Ct values were calculated by the Mx4000 software based on fluorescence intensity values after normalization with an internal reference dye and baseline correction. Statistical analysis of the real-time data were done using a comparison (z-test of the difference between two means) of the corrected Ct means for the three specimens in the proliferative and three specimens in the secretory phases for each gene (26).
In situ hybridization
Out of thirteen different genes studied by RT-PCR and real-time PCR, five genes were randomly chosen for in situ hybridization. In situ hybridization of endometrial frozen sections (three in the mid-late proliferative and three in the mid-secretory phase endometrium) was conducted with 35S-UTP-labeled (DuPont NEN Life Science Products, Boston, MA) sense and antisense riboprobes for: Wnt-7a (ligand), Fz-6 (receptor), FrpHE, and Dkk-1 (inhibitors) and ß-catenin (downstream modulator), as described previously (27) with minor modifications. Briefly, 10-µm frozen sections of endometrium were fixed in 4% paraformaldehyde in PBS, treated with pronase E (0.125 µg/µl) in 50 mM Tris (pH 7.5) and 5 mM EDTA, and postfixed in 4% paraformaldehyde. Then the endometrial sections were acetylated with 0.25% acetic anhydride/0.1 M triethanolamine (pH 8.0), dehydrated through an ascending series of alcohols, and were incubated at 55 C overnight (16 h) in the hybridization solution containing the appropriate concentration of the sense and antisense probe (105 cpm/µl). After hybridization all slides were treated with RNase A, washed, and dehydrated in an ascending series of alcohols. The slides were coated with NTB2 autoradiographic emulsion (Eastman Kodak, Rochester, NY), stored at 4 C for 10 d, developed in D-19 (Eastman Kodak), and lightly counterstained with hematoxylin.
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Results |
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RT-PCR experiments using specific primers (Table 1
in Materials and Methods) demonstrate (Fig. 1) the expression of Wnt-2, Wnt-3, Wnt-4, Wnt-5a, Wnt-7a, Wnt-8b, Fz-6, LRP-6, FrpHE, Dkk-1, Dvl-1, GSK-3ß, and ß-catenin in human endometrium. Wnt-2, Wnt-4, Wnt-5a, Wnt-7a, GSK-3ß, ß-catenin, LRP-6, Fz-6, and Dvl-1 were equally expressed in proliferative and secretory phase endometrium. However, Dkk-1 expression was dramatically increased during the secretory phase, and there was consistent increase in FrpHE expression in most of the samples during the proliferative phase (Fig. 1). Placenta was used as a positive control for Wnt-2, Wnt-3, Wnt-4, Wnt-5a, Wnt-7a, GSK-3ß, ß-catenin, LRP-6, Fz-6, and Dvl-1 and as a negative control for FrpHE. The integrity and relative amounts of these mRNAs were confirmed using GAPDH as a constitutively expressed marker.
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Regulation of expression of Wnt family members in human endometrium in the proliferative and secretory phases was quantified using real-time RT-PCR. The data confirmed that all studied Wnt members are expressed in human endometrium. Specificity of each RT-PCR product was documented using gel electrophoresis (data not shown) and resulted in a single product with appropriate length (Table 2 in Materials and Methods). Melting curve analysis was performed after every run and resulted in a single product for each target gene. Mean Ct values (n = 3 for mid- to late proliferative phase and n = 3 for mid-secretory phase run in quadruplicates) with corresponding SDs are shown in Table 3.
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).
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Wnt-7a is expressed exclusively by luminal epithelial cells in human endometrium.
In situ hybridization data (Fig. 3, A–D), using a 411-bp antisense riboprobe for the Wnt-7a cDNA fragment, reveal localization of Wnt-7a expression exclusively to the luminal epithelium of the endometrium. Wnt-7a is not expressed by glandular epithelium or stroma in normal human endometrium, and there is no noticeable difference in expression between proliferative and secretory endometrium. The sense probe did not show any signal in any of the cell types (data not shown).
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, E–H), consistent with the RT-PCR and quantitative PCR data. The sense probe did not show any specific signal (data not shown).
ß-Catenin, Dkk-1, and Fz-6 expression.
ß-Catenin was found to be expressed in glandular, as well as stromal cells, and is abundant during both the proliferative and secretory phases of endometrial cycle (Fig. 3, I–L), consistent with the PCR data. Dkk-1 was highly expressed by stromal cells in the secretory phase of the cycle, with no or little expression in the proliferative phase, also consistent with the PCR data. Low expression of Dkk-1 was found in glandular cells (Fig. 3, M–P). Frizzled-6 expression is predominantly located in glands, compared with stromal cells, in both proliferative and secretory endometrium. There was no appreciable difference between the proliferative and secretory phases (Fig. 3, Q–T). No signal was observed with the sense probes for all the studied genes (data not shown).
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Discussion |
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We did not find significant regulation of Wnt-2, Wnt-4, Wnt-5a, and Wnt-7a mRNA expression in proliferative and secretory phase endometrium, which is consistent with another study in human endometrium (23). In contrast, we observed significant up-regulation of Wnt-3 (4.7 fold, P < 0.05) in proliferative endometrium that was not found in a previous study (23), likely due to different experimental methods for detection with different sensitivities. Some Wnt ligands are involved in reproductive processes and participate in mesenchymal-epithelial interactions in mouse endometrium. Wnt-4 is crucial for sex-specific development of the female reproductive tract (30). Wnt-5a is expressed in the uterine mesenchyme but not in the epithelium, and tissue recombinant experiments demonstrate that expression of the homeobox-containing gene Msx1 in the epithelium depends on expression of Wnt-5a in the underlying mesenchyme of uterine origin (31). In turn, Wnt-5a expression in the stroma is maintained by expression of Wnt-7a in the luminal epithelium. These studies together indicate that Wnt signaling plays important roles in mediating mesenchymal-epithelial interactions in the endometrium. Furthermore, Msx1 is highly expressed in mouse uterine epithelial cells that undergo pronounced changes in morphology in response to embryo implantation during early pregnancy, suggesting a possible role of Wnt signaling in this process. Whether these mechanisms are operational in human endometrium is currently under investigation in our laboratory.
Reports on the expression and regulation of Wnt receptors and downstream effectors are very limited in reproductive tissues, and to our knowledge this is the first study on the pattern of mRNA expression and regulation of these molecules in the endometrium. There was no significant change in the expression of studied Wnt receptors (Fz-6 and LRP-6) and the downstream effectors (Dvl-1, GSK-3ß, and ß-catenin) in proliferative and secretory phase human endometrium, suggesting that their expression is not directly regulated by ovarian steroid hormones in this tissue. ß-catenin, which can be found in the plasma membrane, in the cytoplasm and the nucleus, is a key mediator of Wnt signaling and has an important role as a cell-cell adhesion molecule allowing association of cadherins with gap junction proteins (32). In the cytoplasm, ß-catenin can associate with the adenomatous polyposis coli-axin-complex, leading to its degradation (33), whereas the free, unbound, and nonphosphorylated form of ß-catenin enters the nucleus and activates Wnt target genes. Our data showing no statistically significant difference in ß-catenin expression between the proliferative and secretory phase of the cycle are consistent with the status of ß-catenin phosphorylation, rather than the intracellular protein levels per se, as the key variable in activating downstream transcription (34), although mechanistically the actions of Wnt signaling and ß-catenin in endometrial function remain to be elucidated.
Inhibitors of the Wnt signaling pathway are important in regulating Wnt actions (5, 6). Our results are in agreement with those from a previous study (35), and studies on global gene profiling of human endometrium from our laboratory (3) and others (2, 4) that indicate dramatic changes in expression of FrpHE and Dkk-1 during the cycle. In the current study, there was 22.2-fold down-regulation of FrpHE and 234.3 fold up-regulation of Dkk-1 in the secretory phase (Fig. 2), suggesting that progesterone stimulates Dkk-1 expression while inhibiting FrpHE expression in human endometrium and/or that estrogen up-regulates FrpHE. Supporting this are observations that FrpHE is markedly up-regulated in estrogen-dependent endometrial and breast carcinomas, but not other cancers (35). These observations are consistent with a role for FrpHE in endometrial proliferation and overexpression with neoplastic development in estrogen-responsive tissues. Down-regulation of FrpHE and up-regulation of Dkk-1 in the secretory phase are associated with concomitant endometrial differentiation and preparing the endometrium for receptivity to embryonic implantation. Recent studies suggest that Dkk-1 interacts with the coreceptors LRP5/6 and inhibits Wnt signaling by disrupting binding of LRP5/6 to the Wnt/Fz ligand-receptor complex (16) and that FrpHE inhibits Wnt action by competitive binding to Wnt ligand(s) (15). However, the precise mechanisms of how the two inhibitors differentially regulate endometrial function, as well as mechanisms underlying Wnt regulation and action in human endometrium, are not well understood and are currently under investigation in our laboratory.
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Footnotes |
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Abbreviations: Ct, Threshold cycle; Dkk, Dickkopf; Dvl, Dishevelled; Fz, frizzled; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GOI, gene of interest; GSK, glycogen synthase kinase; LRP, low-density lipoprotein receptor-related protein; RT, reverse transcription.
Received March 21, 2003.
Accepted May 12, 2003.
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