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Research Article |
School of Anatomy and Human Biology, The University of Western Australia, Perth, Western Australia 6009, Australia
ABSTRACT
Wnt genes regulate a diverse range of developmental processes, including placental formation. Activation of the WNT pathway results in translocation of beta-catenin (CTNNB1) into the nucleus and the subsequent activation of transcription factors that promote proliferation. The secreted frizzled related proteins (SFRPs) are thought to inhibit WNT signaling by binding to the WNT ligand or its frizzled receptor. In this study, we compared the expression patterns of one of these secreted molecules, SFRP4, in the two morphologically and functionally distinct regions of the rat placenta during the last third of pregnancy. In addition, we assessed whether placental SFRP4 expression is altered in a model of glucocorticoid-induced placental growth restriction. Temporal analyses of the rat placenta by quantitative RT-PCR, in situ hybridization, and immunohistochemistry during the final third of pregnancy demonstrated elevated levels of Sfrp4 mRNA and SFRP4 protein near term, specifically in trophoblast cells of the basal zone. This increase in expression of SFRP4 in basal zone trophoblasts was associated with a reduction in CTNNB1 nuclear translocation, consistent with inhibition of the WNT pathway. Maternal dexamethasone treatment (1 µg/ml of drinking water, Days 13–22), which has previously been shown to reduce placental growth, further increased the expression of Sfrp4 mRNA in both the basal and labyrinth zones of the placenta at Day 22. Collectively, these data demonstrate that increased expression of SFRP4 is associated with reduced growth of placental regions in normal pregnancy and after glucocorticoid-induced growth retardation. These observations, together with associated changes in CTNNB1 localization, support the hypothesis that increased placental expression of SFRP4 inhibits the WNT pathway and thereby influences placental growth via effects on cell fate signaling.
apoptosis, b-catenin, glucocorticoid, growth retardation, placenta, syncytiotrophoblast, trophoblast, SFRP4, WNT
INTRODUCTION
Intrauterine growth restriction (IUGR) is a major health concern because it increases the rate of neonatal morbidity and mortality [1] and the subsequent incidence of adult-onset diseases due to fetal programming [2]. The control of fetal growth involves a range of genetic and environmental factors that are mediated, in part, via the placenta [3, 4]. Thus, while a strong relationship between placental function and fetal well-being is well recognized [5–8], the complex regulation of trophoblast proliferation and apoptosis remains poorly understood. Gene deletion studies have established a critical role for WNT signaling in placental development [9–12]; thus, modulation of WNT action may affect proliferation and apoptosis of placental trophoblasts and consequently fetal growth.
WNT signaling is initiated by binding of WNT ligands to a transmembrane frizzled (FZD)/lipoprotein receptor–related protein complex resulting in the activation of the cytoplasmic protein dishevelled (DVL). DVL inactivates glycogen synthase kinase-3ß (GSK3B), which results in the accumulation and subsequent nuclear translocation of ß-catenin (CTNNB1). In the presence of appropriate coactivators, CTNNB1 binds the T-cell factor/lymphoid enhancer–binding factor (TCF/LEF) family of DNA-binding proteins and upregulates transcription of various target genes that drive proliferation and cell fate determination. In the absence of WNT signaling, the activated GSK3B forms an intracellular complex with adenomatous polyposis coli and axin, targeting CTNNB1 for degradation via the ubiquitin-proteosome pathway removing the trophic stimulus [13–15].
WNT signaling has been previously demonstrated in human cytotrophoblasts [16], and recent investigations demonstrate a critical role for the WNT pathway in placental development. Specifically, Wnt7b and the Tcf/Lef1 genes are required for normal chorioallantoic fusion [9, 12], and Wnt2 null mice display impaired placental angiogenesis, reduced birth weight, and a higher rate of mortality [11]. Moreover, Fzd5 knockout mice display defects in placental vascularization, implicating a role for WNT5A and WNT10B, the physiological ligands of FZD5, in placental development [10]. A role for CTNNB1, the key mediator of WNT signaling, has also been implicated in trophoblast adhesion [17], survival [18], and differentiation [19].
Secreted frizzled related proteins (SFRPs) are structurally similar to the frizzled receptors but lack the transmembrane and intracellular components necessary for signal transduction [20]. It is proposed that SFRPs inhibit the WNT pathway by binding to either the WNT ligand or frizzled receptor [21, 22], potentially inhibiting the entire canonical pathway [23]. Inhibition of the WNT pathway results in continual degradation of CTNNB1 and the absence of TCF/LEF-mediated gene expression. Previous data have established a role for one of these secreted proteins, SFRP4, in involution of reproductive tissues, possibly due to a proapoptotic action [24–26] and in apoptosis of thecal cells during ovulation [27, 28]. Furthermore, levels of SFRP4 expression inversely correlate with tumor progression in human endometrial stromal and undifferentiated endometrial sarcomas [29]. Thus, placental expression of SFRP4 is likely to impact placental growth via effects on the balance between cellular proliferation and apoptosis.
The present study, therefore, investigated the placental expression and distribution of SFRP4 and CTNNB1 during the final third of pregnancy, the period of maximal fetal and placental growth. Separate analyses were made of the two functionally and morphologically distinct zones of the rat placenta, the basal and labyrinth zones, because the latter grows extensively between Day 16 and Day 22 [30, 31], whereas the basal zone does not grow and undergoes a notable increase in apoptosis during the same period [32]. These analyses showed marked upregulation of Sfrp4 mRNA in the basal zone near term but not in the rapidly growing labyrinth zone. Therefore, because glucocorticoids can induce placental growth restriction and trophoblast apoptosis [31–34] and are known to inhibit WNT signaling in other cell types [35], we also tested the hypothesis that maternal dexamethasone treatment stimulates placental SFRP4.
MATERIALS AND METHODS
Animals
Nulliparous albino Wistar rats aged between 8 and 12 wk were obtained from Animal Resources Centre (Murdoch, Australia) and maintained under controlled conditions as described previously [30]. Rats were mated overnight, and the day in which spermatozoa were present in a vaginal smear was designated Day 1 of pregnancy. All procedures involving animals were conducted after approval by the Animal Ethics Committee of The University of Western Australia.
Glucocorticoid Treatment
Increased fetal and placental glucocorticoid exposure was achieved by administration of dexamethasone acetate (1 µg/ml; Sigma, St. Louis, MO) in the drinking water from Day 13 to Day 22 of pregnancy (term, 23 days). It has previously been shown that this treatment reduces fetal and placental weights at Day 22 by 25%–30% [31, 32].
Tissue Collection
For collection of placental zones, pregnant rats were anesthetized with halothane/nitrous oxide at either Day 16 or Day 22 of gestation, and three fetuses and placentas were removed from each mother. Placental zones were separated by blunt dissection and snap frozen in liquid nitrogen for real-time quantitative RT-PCR, or alternatively whole placentas were fixed in Histochoice MB (Amresco, Solon, OH) for immunohistochemistry.
RNA Sample Preparation
Total RNA was isolated from placental zones at Day 16 and Day 22 of pregnancy using Tri-Reagent (Molecular Resources Center, Cincinnati, OH) as per the manufacturer's instructions. RNA integrity was assessed by ethidium bromide staining of the nucleic acids before agarose gel electrophoresis (data not shown). Total RNA was treated to remove contaminating genomic DNA using DNA-free reagent (Ambion, Austin, TX). Deoxyribonuclease-treated total RNA (1 µg) was used to synthesize cDNA using M-MLV Reverse Transcriptase RNase H Point Mutant and random hexamer primers (Promega, Madison, WI) as per the manufacturer's instructions. The resultant cDNAs were purified using the Ultraclean PCR Cleanup kit (MoBio Industries, Solana Beach, CA).
Real-time RT-PCR
Analyses of expression levels for Sfrp4, Ctnnb1, and Rpl19 transcripts were performed by real-time RT-PCR on the Rotorgene 3000 (Corbett Industries, Sydney, Australia) using iQ SYBR Green Supermix (BioRad, Hercules, CA). Primers for Sfrp4 and Ctnnb1 (Table) were designed using Primer 3 software (MIT/Whitehead Institute, http://www-genome.wi.mit.edu) [36]. Each of the selected primer pairs was positioned to span introns to ensure no product was amplified from genomic DNA, and the resulting amplicons were sequenced to confirm specificity (data not shown). All samples were standardized against Rpl19 as previously described [37]. Standard curves for each product were generated from gel-extracted (QIAEX II; Qiagen, Melbourne, Australia) PCR products using 10-fold serial dilutions and the Rotorgene 3000 software.
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Immunohistochemistry
Sections of placenta were cut at 4 µm, deparaffinized and rehydrated, and then incubated for 10 min in 3% H2O2 to block endogenous peroxidases. Mouse monoclonal primary antibody to CTNNB1 (C7082, Sigma) and rabbit polyclonal primary antibody to SFRP4 (1:50; a gift from Dr. Robert Friis, University of Bern, Bern, Switzerland) were each applied overnight at 4°C followed by a 30-min room temperature incubation in biotinylated anti-mouse or anti-rabbit secondary at 1:200 (BA2001 and BA1000; Vector Laboratories, Burlingame, CA) and streptavidin-peroxidase complex at 1:50 (Vectastain ABC Kit, Vector Laboratories). Positive staining was visualized by application of diaminobenzidine substrate (Sigma) and counterstained with hematoxylin before dehydration in graded alcohols and mounting in DPX.
In Situ Hybridization
In situ hybridization with a digoxigenin-labeled riboprobe to localize the expression of Sfrp4 mRNA was performed as described previously [21, 25, 27]. Briefly, 5-µm sections were dewaxed and rehydrated in PBS before pretreatment with proteinase K (10 mg/ml) for 15 min at 37°C in a humidifying chamber. Slides were acetylated with fresh acetic anhydride followed by incubation in prehybridization solution for 2 h at 55°C and hybridization in 200 ng/ml of labeled sense or antisense probe (16 h at 55°C). The following day, the slides were washed extensively, then blocked (0.5% w/v blocking agent in Tris-buffered saline) before addition of antidigoxigenin antibody (1:1000) for 1 h at room temperature. Sfrp4 mRNA was visualized by addition of nitro blue tetrazolium/5-bromo,4-chloro,3-indolyphosphate solution (20 µl/ml) and incubation in darkness until sufficient contrast was obtained, halting the color reaction with distilled water.
Statistical Analysis
All data are expressed as means ± SEM. Two-way ANOVA (GenStat7, Hemel Hempstead, United Kingdom) was used to assess variation in expression levels of Sfrp4 and Ctnnb1 mRNA (standardized to Rpl19 expression) for gestational age and placental zone. For comparisons in which the F test for the ANOVA reached statistical significance (P < 0.05), differences were assessed by Student t-test [38].
RESULTS
Expression of Sfrp4 in the Rat Placenta During Normal Pregnancy
The mRNA for Sfrp4 was readily detectable by real-time RT-PCR in both zones of the placenta (Fig. 1A). Expression of Sfrp4 mRNA increased in the basal zone during the final third of pregnancy (14-fold from Day 16 to Day 22, P < 0.01) and was markedly higher in the basal compared with the labyrinth zone at Day 22 (26-fold, P < 0.001). Localization of Sfrp4 mRNA by in situ hybridization (Fig. 2) reflected the zonal changes in expression observed by RT-PCR and showed that trophoblast cells of the basal zone were the major source of Sfrp4 mRNA expression at Day 22. Weak expression was also observed in the labyrinth zone trophoblast cells near term but not at Day 16.
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Immunohistochemical analysis detected the presence of the SFRP4 protein in the basal zone trophoblast, with minimal expression in the labyrinth zone (Fig. 3, A and B). The SFRP4 protein at Day 16 was demonstrated to be weakly expressed throughout all cell types of the placenta, consistent with mRNA distribution obtained by real-time RT-PCR analysis, and its intracellular distribution was primarily cytoplasmic (Fig. 3B). By Day 22, however, expression of the SFRP4 protein was exclusively limited to the cell membrane or extracellular space surrounding basal zone trophoblasts (Fig. 3, C and D).
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Expression of CTNNB1 in the Rat Placenta During Normal Pregnancy
The expression and distribution of CTNNB1 was used as an end point of WNT signaling and thus the functional capacity of the SFRP4 protein. Real-time RT-PCR analysis demonstrated that the expression of Ctnnb1 mRNA did not change in either zone between Day 16 and Day 22 (Fig. 1B). There was, however, 3-fold higher expression of Ctnnb1 mRNA in the labyrinth compared with the basal zone of Day 22 placentas (P < 0.001).
Expression of the CTNNB1 protein at Day 16 was localized primarily to the nuclei of basal zone trophoblast cells, with weak cytoplasmic expression (Fig. 4, A and B). At Day 22, while most cells still exhibited nuclear expression of CTNNB1, there were now substantial numbers that were entirely CTNNB1 negative (Fig. 4, C and D). Notably, intense nuclear, cytoplasmic, and membranous staining of CTNNB1 was also observed in the intraplacental yolk sac of the labyrinth zone near term (Fig. 5).
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Expression of SFRP4 and CTNNB1 after Maternal Dexamethasone Treatment
Maternal dexamethasone treatment resulted in increased expression of Sfrp4 mRNA at Day 22 (1.2-fold and 2.8-fold higher in the basal and labyrinth zones, respectively, P < 0.05; Fig. 6A). The expression of Sfrp4 was 15-fold higher (P < 0.001) in the basal zone compared with the labyrinth zone of dexamethasone-treated mothers, consistent with the pattern of expression observed in control placentas.
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In situ hybridization for Sfrp4 and immunohistochemistry for SFRP4 demonstrated patterns of placental distribution similar to those observed in the untreated controls. Sfrp4 mRNA was primarily localized to the basal zone trophoblast cells (Fig. 7), whereas SFRP4 protein was localized to the cell membrane and the extracellular space around basal zone trophoblasts (Fig. 8, A and B).
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Real-time RT-PCR analysis of Ctnnb1 mRNA revealed reduced expression in the labyrinth zone of placentas from dexamethasone-treated mothers (42%, P < 0.01; Fig. 6B). No change was observed in Ctnnb1 mRNA expression in the basal zone of dexamethasone-treated placentas. Intracellular distribution of the CTNNB1 protein was unchanged by dexamethasone treatment as determined by immunohistochemistry (Fig. 8, C and D).
DISCUSSION
This study investigated the expression of SFRP4 and CTNNB1, both key regulators of cellular proliferation and apoptosis, in the basal and labyrinth zones of the rat placenta during the period of maximal fetal and placental growth. Sfrp4 mRNA expression increased markedly in basal zone trophoblasts near term but remained minimal in the rapidly growing labyrinth zone. The elevation in basal zone expression of Sfrp4 mRNA was associated with increased localization of the SFRP4 protein to the extracellular space and plasma membrane of these cells. During the same period, the expression of Ctnnb1 mRNA remained unchanged, but nuclear localization of the CTNNB1 protein was reduced, consistent with the absence of basal zone growth. Maternal dexamethasone treatment increased expression of SFRP4 in both the basal and labyrinth zones, an effect that may contribute to the reduction in placental growth [31] and the increase in apoptosis previously observed in this model [32]. Collectively, these data are consistent with inhibition of the WNT pathway by SFRP4, with downstream effects on cell fate signaling that affect placental proliferation and apoptosis.
The control of placental growth involves a delicate balance of proliferative and apoptotic signaling. In the present study, we demonstrate that SFRP4 expression increases markedly in the nonproliferative basal zone near term but remains very low in the rapidly growing labyrinth zone. Moreover, SFRP4 expression was also stimulated by dexamethasone in the basal and labyrinth zones, both of which exhibit reduced growth in this model [31]. These observations are consistent with the antiproliferative action of SFRP4 in mesothelioma [39] and in uterine [29] and colorectal [23] cancer and support the concept that glucocorticoid-mediated inhibition of the WNT pathway [35] may involve, in part, increased SFRP4 expression. Previous studies have also linked expression of SFRP4 to increased apoptosis in a range of reproductive tissues [24–27, 40], and so the rise in SFRP4 specifically within the basal zone between Day 16 and Day 22 of pregnancy is in accord with the previous observation that only the basal zone shows increased apoptosis during this period [32]. Fujita et al. [41] demonstrated increased expression of Sfrp4 mRNA corresponding with decidual cell proliferation between Day 9 and Day 15 of rat pregnancy. This suggests that SFRP4 may affect proliferation and apoptosis in a tissue-specific manner, but further studies are required to assess this possibility.
The function of the frizzled related proteins is thought to involve inhibition of the canonical WNT pathway via competitive binding to the WNT ligand or the transmembrane frizzled receptor [22]. The membranous and extracellular distribution of the SFRP4 protein in the basal zone of the rat placenta observed in this study is consistent with its binding to either the frizzled receptor or the WNT ligand. A similar distribution was recently noted for SFRP4 in prostate cancer in association with reduced rates of proliferation [42], and a loss of membranous SFRP4 expression occurs in the transition from normal esophageal epithelium to esophageal adenocarcinoma [43]. In contrast, the SFRP4 protein has also been localized to the cytoplasm of a colorectal cancer cell line [44] in association with increased proliferation. Collectively, these data indicate that the subcellular localization of SFRP4 may determine its major function, and in the case of the basal zone trophoblast, the membranous localization of SFRP4 suggests that it inhibits the WNT pathway.
The effects of WNT pathway inhibition by frizzled related proteins, including SFRP4, are likely to be mediated via effects on CTNNB1 localization [21, 22]. Thus, the canonical WNT pathway promotes proliferation via the nuclear accumulation of CTNNB1 and subsequent activation of the TCF/LEF family of transcription factors [13, 14] and has been implicated specifically in trophoblast differentiation [17, 19] and survival [18]. The present study shows that increased expression of SFRP4 in basal zone trophoblasts is accompanied by reduced CTNNB1 nuclear localization, consistent with inhibition of the WNT pathway by SFRP4. The association between the expression of SFRP4 and CTNNB1 and the relative amounts of proliferation and apoptosis, however, is still unclear. Feng Han et al. [44] found increased expression of both SFRP4 and CTNNB1 in human colorectal carcinoma, suggesting that SFRP4 may not inhibit the nuclear accumulation of CTNNB1. Similarly, recent reports on the expression of SFRP1, SFRP2, SFRP4, and SFRP5 indicate that, of the isoforms examined, only SFRP4 did not reduce CTNNB1 nuclear expression or induce apoptosis in a colorectal cancer cell line [23]. In the same model, however, SFRP4 reduced colony formation [23], and so the SFRP4-associated reduction in CTNNB1 observed in the basal zone trophoblasts may relate primarily to an inhibition of proliferation and induction of apoptosis. Further in vitro studies are required to confirm a causal link between SFRP4 action and inhibition of WNT-mediated trophoblast proliferation.
Normal placental development requires rapid growth and vascularization of maternal and fetal blood spaces, whereas in IUGR this process is compromised. Zhang et al. [45] proposed that the WNT pathway may play a role in promoting angiogenesis, specifically by upregulation of vascular endothelial growth factor (VEGF) expression in colonic epithelial cells. Therefore, the upregulation of SFRP4 following dexamethasone treatment may result in inhibition of the WNT pathway and a subsequent reduction in VEGF expression in the labyrinth zone. In recent preliminary studies, we observed that dexamethasone markedly reduces the expression of Vegf mRNA in the placental labyrinth zone (Hewitt, Mark, and Waddell, unpublished data). Moreover, gene deletion studies involving various components of the WNT pathway [10–12] all show defective placentation, particularly in relation to angiogenesis, suggesting that vascular development of the placenta is reliant on the WNT signaling pathway.
The distribution of CTNNB1 within the placental labyrinth was predominantly within the nucleus and cytoplasm of trophoblast cells lining the intraplacental yolk sac, an extension of the yolk sac into the labyrinth zone [46] known to play a role in maternal-fetal calcium transfer [47, 48]. Although CTNNB1 may affect cell fate signaling in these cells via the WNT pathway as already discussed, its membranous localization suggests that it may also play a role in cell-cell adhesion and migration as previously demonstrated in various cell types [15]. Whatever the function of CTNNB1 in the labyrinth zone, it is likely to have been compromised by dexamethasone treatment, which reduced the expression of Ctnnb1 mRNA, specifically in the labyrinth zone at Day 22.
In conclusion, this study shows that expression of SFRP4 increases markedly near term in the nonproliferative placental basal zone but not in the rapidly growing labyrinth zone. Associated changes in CTNNB1 localization suggest that SFRP4 inhibits the WNT pathway in trophoblast cells. Dexamethasone treatment further increased placental SFRP4 expression at term, consistent with the hypothesis that SFRP4 plays an antiproliferative and/or proapoptotic role in glucocorticoid-induced placental growth restriction.
FOOTNOTES
1 Correspondence: Brendan J. Waddell, School of Anatomy and Human Biology, The University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia. FAX: 61 8 6488 1051; bwaddell{at}anhb.uwa.edu.au 
Received: 20 September 2005.
First decision: 7 November 2005.
Accepted: 9 March 2006.
REFERENCES
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