REVIEW ARTICLE published: 26 September 2013 doi: 10.3389/fgene.2013.00190 Human placental invasion and differentiation: a particular focus on Wnt signaling

Martin Knöfler* and Jürgen Pollheimer Department of Obstetrics and Fetal-Maternal Medicine, Reproductive Biology Unit, Medical University of Vienna, Austria

Edited by: Wingless ligands, a family of secreted proteins, are critically involved in organ development Marie Van Dijk, VU University Medical and tissue homeostasis by ensuring balanced rates of stem cell proliferation, cell death Center, Netherlands and differentiation. Wnt signaling components also play crucial roles in murine placental Reviewed by: development controlling trophoblast lineage determination, chorioallantoic fusion and Sascha Drewlo, Wayne State University School of Medicine, USA placental branching morphogenesis. However, the role of the pathway in human placen- Nardhy Gomez-Lopez, Wayne State tation, trophoblast development and differentiation is only partly understood. Here, we University, USA summarize our present knowledge about Wnt signaling in the human and discuss *Correspondence: its potential role in physiological and aberrant trophoblast invasion, gestational diseases and Martin Knöfler, Department of choriocarcinoma formation. Differentiation of proliferative first trimester Obstetrics and Fetal-Maternal Medicine, Reproductive Biology Unit, into invasive extravillous is associated with nuclear recruitment of β-catenin Medical University of Vienna, and induction ofWnt-dependentT-cellfactor 4 suggesting that canonicalWnt signaling could Waehringer Guertel 18-20, A-1090 be important for the formation and function of extravillous trophoblasts. Indeed, activation Vienna, Austria e-mail: martin.knoefler@ of the pathway was shown to promote trophoblast invasion in different in vitro trophoblast meduniwien.ac.at model systems as well as trophoblast cell fusion. Methylation-mediated silencing of inhibitors of Wnt signaling provided evidence for epigenetic activation of the pathway in placental tissues and choriocarcinoma cells. Similarly, abundant nuclear expression of β-catenin in invasive trophoblasts of complete hydatidiform moles suggested a role for hyper-activated Wnt signaling. In contrast, upregulation of Wnt inhibitors was noticed in placentae of women with preeclampsia, a disease characterized by shallow trophoblast View metadata, citation and similar papers at core.ac.uk brought to you by CORE invasion and incomplete spiral artery remodeling. Moreover, changes in Wnt signaling have been observed upon cytomegalovirus infection and in recurrent abortions. In summary, provided by Frontiers - Publisher Connector the current literature suggests a critical role of Wnt signaling in physiological and abnormal trophoblast function.

Keywords: placenta, human, trophoblast, invasion,Wnt

DEVELOPMENT AND FUNCTION OF THE HUMAN PLACENTA covering the surface of floating villi are con- Development of the human placenta is critical for embryonic tinuously generated by asymmetrical cell division, differentiation development and successful pregnancy outcome. Immediately and fusion of villous cytotrophoblasts (vCTBs) with the develop- after implantation, trophectodermal cells forming the outermost ing syncytium (Aplin, 2010). The latter also secretes numerous epithelial layer of the blastocyst give rise to diverse trophoblast cell hormones, such as human chorionic gonadotrophin, into the types (Hamilton and Boyd, 1960; Cross et al., 1994). Cell fusion maternal circulation, which are required for maintenance and generates the primitive syncytium underneath the implanted immunological adaptation of pregnancy (Bansal et al., 2012). embryo, which may represent the earliest invasive trophoblast cell Fusion process and numbers of vCTBs decrease during preg- type migrating into the maternal endometrium (Figure 1A). After nancy. Hence at term, syncytiotrophoblasts are in close contact formation of the lacuna system, the ancestor of the intervillous with placental vessels allowing efficient nutrient uptake by the space, cytotrophoblasts (CTBs) emanating from the trophecto- fetus. dermal layer generate primary villi by proliferation and invasion Villi connected to the basal plate of the human placenta give through the primitive syncytium (Figure 1B). Throughout preg- rise to proliferative cell columns from which differentiated extrav- nancy, these primary villi transform into secondary and tertiary illous trophoblast (EVT) cell types are generated. At early stages of villi characterized by invasion of extraembryonic mesenchymal pregnancy, invasive endovascular cytotrophoblasts (eCTBs) plug cells, villous branching, and vascularization. During the first the maternal arterioles to prevent premature onset of blood flow trimester of pregnancy two types of mature villi can be discrimi- into the intervillous space (Pijnenborg et al., 2006). Failures in this nated, which are floating and anchoring villi (Figure 1C). Floating process were shown to be associated with pregnancy complications villi connected to the intervillous space represent the transport such as abortions likely due to the premature rise in oxygen lev- units of human placenta. After establishment of blood flow els which may provoke oxidative stress and damage of placental nutrients and oxygen pass the epithelial layers of these villi ensur- villi (Hustin et al., 1990; Burton et al., 2010). Besides endovascular ing appropriate fetal development and growth. Multinucleated invasion, interstitial cytotrophoblasts (iCTBs) migrate into the

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FIGURE 1 | Critical steps of human placental development. (A) After vessels from outside and eventually form giant cells as the end stage of implantation stems cells of the trophectoderm give rise to the primitive the invasive differentiation pathway. Endovascular trophoblasts migrate syncytium by cell fusion. In this region lacunae, the ancestor of the into spiral arteries and contribute to uNK cell-initiated remodeling within intervillous space, are formed. Some of the lacunae erode uterine the and the upper part of the myometrium. AE, amniotic vessels. (B) At a subsequent stage, proliferative cytotrophoblasts (CTBs) epithelium; CCT, cell column trophoblast; DF, decidual fibroblast; EB, emanate from the trophectoderm, break through the primitive syncytium embryoblast; EM, extraembryonic mesoderm; eCTB, endovascular and contact the basal plate thereby forming primary villi. (C) Tertiary villi ; GC, giant cell; ICM, inner cell mass, iCTB, interstitial are built upon migration of extraembryonic mesodermal cells into the cytotrophoblast; LUE, luminal uterine epithelium; L, lacunae, pF, placental primary structures and vascularization. At distal sites, proliferative cell fibroblast; PS, primitive syncytium; pV, placental vessel; SA, spiral artery; columns are formed which give rise to different invasive extravillous S, syncytium; TE, trophectoderm; UG, uterine gland; uNK, uterine NK cell; trophoblast subtypes. iCTBs migrate into decidual stroma approach UV, uterine vessel; vCTB, villous cytotrophoblast. maternal decidua likely cross-talking to diverse uterine cell types, 2010). In particular, interaction of paternal human leukocyte including uterine natural killer cells, macrophages, and decid- antigen (HLA)-C expressed on iCTBs with maternal killer cell ual stromal cells (Bulmer et al., 2010; Oreshkova et al., 2012). immunoglobulin-like receptors (KIR) are thought to play a piv- These interactions are thought to be important for immunolog- otal role in and reproductive success (Hiby et al., ical acceptance of the placental/fetal allograft as well as for the 2004). In the absence of blood flow, trophoblast invasion might timing and depth of trophoblast invasion (Redman and Sargent, be controlled by factors secreted from uterine glands, such as

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epidermal growth factor (EGF) and vascular endothelial growth into EVTs(Caniggia et al.,2000b; van Dijk and Oudejans,2011). In factor (vEGF), which are also thought to be crucial for pla- contrast, AP-2α, signal transducer and activator of transcription 3 cental and embryonic growth at this early stage of pregnancy (STAT3)or glial cells missing 1 (GCM1) promote trophoblast inva- (Burton et al., 2007). With the establishment of the maternal- sion and GCM1 was also shown to inhibit proliferation of vCTBs placental circulation, the placenta switches from histiotrophic (Poehlmann et al., 2005; Baczyk et al., 2009; Biadasiewicz et al., to haemotrophic nutrition around the 12th week of gestation, 2011). Hence, it is assumed that a set of key regulatory transcrip- plugging of vessels is dissolved and extensive remodeling occurs. tion factors controls the switch between trophoblast proliferation Transformation of the maternal spiral arteries into large diame- and EVT differentiation (Loregger et al., 2003; Knöfler and Poll- ter vessels likely ensures adapted nutrient supply, reduced vessel heimer, 2012). EVT formation is accompanied by the expression contractility and constant oxygen delivery at low blood pressure of distinct intergrins which are induced in a distance-dependent to the developing fetus (Burton et al., 2010). Vessel remodel- manner in vivo as well as in vitro (Damsky et al., 1994; Aplin et al., ing might be initiated by uterine NK cells and both types of 1999). Again, the molecular basis for differentiation-dependent differentiated EVTs are thought to play a crucial role in complet- switching remains unknown. Increasing oxygen concen- ing this process (Robson et al., 2012). Whereas eCTBs displace trations during pregnancy and contact with the decidual matrix maternal endothelial cells and remodel decidual and superficial likely play major roles. Interestingly, accumulating evidence sug- myometrial spiral arteries, iCTBs approach the vessels from out- gests that failures in EVT differentiation could contribute to the side and contribute to elastolysis and disruption of the vascular pathogenesis of pregnancy diseases with restricted trophoblast wall. This involves a series of events such as trophoblast-induced invasion and remodeling. Expression of inhibitor of DNA binding apoptosis of vascular smooth muscle cells (Harris, 2011). Fail- 2 (Id2), blocking the binding activity of differentiation-promoting ures in EVT invasion, remodeling and CTB gene expression basic helix-loop-helix (bHLH) proteins through heterodimerisa- were noticed in different pregnancy diseases such as preeclamp- tion, was shown to be downregulated in EVTsof normal pregnancy sia or severe intrauterine growth restriction (Pijnenborg et al., but maintained in preeclamptic placental tissue (Janatpour et al., 1991, 2006; Zhou et al., 2013). Abnormal contractility and pres- 2000). Along those lines, inhibition of HIF1α-dependent TGFβ3, sure may provoke hypoxia re-oxygenation injuries of floating acting as a negative regulator of trophoblast invasion, restored villi thereby inducing secretion of adverse cytokines and antian- migration in explant cultures of preeclamptic villi emphasizing giogenic molecules such as soluble Fms-like tyrosine kinase-1 the particular role of oxygen in EVT differentiation (Caniggia et al., (sFLT-1) and elevated shedding of micropar- 1999, 2000a). Moreover, upregulation of EVT-specific genes and ticles (Burton et al.,2010; Redman et al.,2012; Tal,2012). The latter invasion were impaired in trophoblasts isolated from preeclamptic are thought to contribute to maternal endothelial cell dysfunction placentae (Lim et al., 1997). Of importance, eCTBs express a char- as part of a global, systemic inflammatory response observed in acteristic pattern of vascular adhesion molecules which, however, preeclampsia. is abnormal in preeclamptic tissues (Zhou et al., 1997a). While the hierarchy and cross-talk of critical molecular events DIFFERENTIATION AND INVASION OF HUMAN controlling EVT differentiation await further investigations, reg- EXTRAVILLOUS TROPHOBLAST ulation of trophoblast invasion has been investigated in a vast Differentiation of proliferative CTBs into growth-arrested EVTs, number of studies using primary cells, choriocarcinoma cells invading decidual tissue and vessels, is thought to involve a series and established non-tumorigenic trophoblast cell lines. The dif- of well-controlled molecular steps which, however, are poorly ferent invasive trophoblast cell types produce sets of proteases, understood. This is partly due to the fact that EVT differentia- i.e., matrix metalloproteinases (MMP), urokinase plasminogen tion in vitro can only be studied upon access to first trimester activator (uPA) and cathepsins, which are thought to degrade placental samples, which in general is limited due to ethical decidual extracellular matrix proteins and thereby facilitate cell considerations. Growth of the trophoblast cell column harbor- invasiveness. The respective inhibitors, tissue inhibitors of met- ing progenitor cells for the invasive differentiation pathway may alloproteinases (TIMPs) and plasminogen activator inhibitors involve paracrine factors released from the underlying placental (PAIs) are produced by EVTs as well as decidual cells to limit such as IGF molecules which also promote prolif- the extent of trophoblast invasion. Numerous soluble factors eration of vCTBs in vitro (Aplin et al., 2000; Forbes et al., 2008). expressed at the fetal-maternal interface including chemokines, Since spontaneous outgrowth and migration in villous explant cytokines and angiogenic factors were shown to promote tro- cultures is achieved in the absence of serum, it is likely that the phoblast motility in an autocrine or paracrine manner (Bischof intrinsic molecular program of the placental villus is also suffi- et al., 2000; Lala and Chakraborty, 2003; Knöfler, 2010).Asacom- cient for the particular differentiation process in vivo.However, mon theme, the secreted proteins were shown to stimulate MMP the precise mechanisms controlling integrity and stability of cell expression and secretion, in particular the gelatinases MMP-2 and columns allowing for balanced rates of growth and differentia- MMP-9. Inhibitory proteins such as TNF, Nodal or TGFβ could tion have not yet been elucidated. Adhesive interactions between restrain trophoblast motility by increasing expression of TIMPs L-selectin expressed in cell columns and its carbohydrate ligands and PAIs (Lala and Graham, 1990; Haider and Knöfler, 2009; could play a role (Prakobphol et al., 2006). Moreover, different Nadeem et al., 2011). Although a complex interplay of growth transcription factors such as oxygen-dependent hypoxia-inducible factors likely controls trophoblast cell migration and invasion, it factor 1α (HIF1α) and Stox1, discussed elsewhere in this issue, are remains unclear whether all of the currently identified effects truly critical for trophoblast cell proliferation and inhibit differentiation play a role in vivo. Tumorigenic and non-tumorigenic cell types as

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well as hybridomas used in functional studies may not accurately diseases including different cancers, diabetes or neurodegenera- mimic trophoblast cell behavior. Indeed, overall gene expression tive disorders (Polakis, 2000; Al-Harthi, 2012). Wnts comprise a profiles of primary CTBs and EVT cultures differ considerably family of palmitoylated, cysteine-rich glycoproteins, which due to from the different established trophoblast cell lines (Bilban et al., their low solubility are secreted in a lipoprotein-bound form or 2010). Moreover, compared to primary cells, a diverging HLA through exosomes (Herr et al., 2012). The first described member profile was identified in the immortalized trophoblast cell lines. of this family of secreted ligands was the Wnt1 proto-oncogene, Villous CTBs lack surface expression of classical HLA molecules, which is homologous to the Drosophila gene Wingless. Originally, but EVTs produce HLA-C, -E, and -G upon differentiation. JEG-3 the gene has been named int-1 since it has been identified as choriocarcinoma cells show a similar HLA profile as EVTs, whereas an integration site for the murine mammary tumor virus which several immortalized cell lines produce HLA-A and -B, suggesting can provoke breast cancer (Nusse et al., 1991). In humans, 19 abnormal activation of these genes during the immortalization different Wnt ligands and 10 seven-transmembrane domain friz- procedure or a non-trophoblastic origin of these cells (Apps et al., zled (Fzd) receptors have been identified (Wodarz and Nusse, 2009). 1998). The latter interact with low density lipoprotein receptor Furthermore, published literature suggests key signaling path- related proteins (LRP-5 or -6) forming a functional, heterodimeric ways that are involved in trophoblast motility. Abundant growth receptor for canonical Wnt signaling. It is likely that the com- factors such as hCG, EGF, HGF, or IGF2 activate MAPK plex interplay of different Wnts with Fzds provokes specific Wnt kinase (MEK)/extracellular regulated kinase (ERK) and phospho- responses depending on the receptor context and the particular inositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin cell type. Stabilization and nuclear recruitment of β-catenin is a (mTOR) signaling, whereas prostaglandins were shown to act hallmark of the canonical pathway. However, Wnt ligands also through the Rho-Rock pathway (Pollheimer and Knöfler, 2005; trigger non-canonical, β-catenin-independent signaling including Knöfler, 2010). Besides expression of TIMPs and PAIs, downreg- the Wnt/Ca2+ and the Wnt/planar cell polarity (PCP) pathway ulation of signaling kinase activity could represent a mechanism (Gordon and Nusse, 2006; Hendrickx and Leyns, 2008; van to limit the extent of trophoblast invasion. For example endo- Amerongen and Nusse, 2009). statin, which could be released from decidual collagen XVIII by EVT-mediated proteolytic cleavage, was shown to impair growth CANONICAL Wnt SIGNALING factor-induced AKT/mTOR phosphorylation and cell migration Canonical Wnt signaling involves a series of steps resulting in the (Pollheimer et al., 2004, 2005, 2011). stabilization and nuclear translocation of β-catenin (Gordon and To identify novel genes and pathways controlling trophoblast Nusse, 2006). In unstimulated cells, β-catenin is predominantly motility and differentiation we and others recently performed found at adherens junctions where it binds to E-cadherin and comparative gene expression studies of CTBs and EVTs isolated α-catenin and thereby maintains epithelial structure and polar- from first trimester placental tissues. Chip-based profiling of ity. Cytosolic levels of β-catenin are low since it is degraded EGFR-positive CTBs, isolated by flow cytometry, and EVTs, gen- in a destruction complex consisting of adenomatous polyposis erated by seeding of the CTBs on fibronectin for 12 h, resulted coli (APC), Axin, casein kinase Iα (CKIα) and glycogen synthase in the identification of 3433 mRNAs which are at least two-fold kinase 3β (GSK-3β). The latter phosphorylates β-catenin at its differentially expressed between the two cell populations (Apps N-terminus and thereby induces binding of β-transducin repeat- et al., 2011). Using immunopurified CTBs and EVTs isolated from containing protein (β-TrCP) and its associated E3 ubiquitin ligase. outgrowths of villous explant cultures and gene chips with a lower This results in ubiquitination and proteasomal degradation of number of probe sets compared to the aforementioned study, we β-catenin (Stamos and Weis, 2013). In contrast, Wnt stimulation detected 991 differentially expressed transcripts in our analyses promotes Fzd-LRP heterodimerisation and cytosolic stabiliza- (Bilban et al., 2009). One of these mRNAs which was found to be tion of β-catenin by disruption of the APC/Axin/GSK-3β/CK1α induced upon EVT differentiation encoded TCF-4, one of the key destruction complex. Upon binding of Wnt to the cysteine-rich transcription factors in Wnt signaling (Roose and Clevers, 1999). domain of Fzd, the multifunctional protein Disheveled (Dvl) is Hence, this result prompted us to investigate the expression pat- recruited to the cytosolic portion of the heterodimeric recep- tern of Wnt signaling components and the general role of the tor and thereby provokes binding of Axin and GSK-3β as well canonical signaling pathway in human trophoblast migration and as GSK-3β-mediated phosphorylation of LRP-5/6 (Mao et al., invasion. 2001; Bilic et al., 2007). This event could either inhibit the catalytic activity of GSK-3β toward β-catenin promoting seques- Wnt SIGNALING PATHWAYS tration or induce internalization and lysosomal degradation of Besides Hippo, Hedgehog, Notch and TGFβ signaling, Wnt sig- components of the destruction complex (Metcalfe and Bienz, naling represents one of the few conserved pathways critically 2011). As a consequence cytoplasmic concentrations of β-catenin involved in developmental processes. From Drosophila to human, increase and active de-phosphorylated β-catenin translocates to the particular signal transduction cascade controls early axis for- the nucleus where it binds to transcription factors of the lymphoid mation, limb patterning and organogenesis (Logan and Nusse, enhancer factor-1 (LEF-1)/TCF family (Clevers, 2006; Gordon 2004; Clevers,2006). In adults,Wnt controls homeostasis of regen- and Nusse, 2006). LEF/TCF proteins are high mobility group erating tissues such by regulating stem cell maintenance, cell fate proteins lacking transcriptional activity and hence require co- decisions and differentiation (Clevers, 2006; Herr et al., 2012). activators or co-repressors for their function. Binding of β-catenin Abnormal Wnt signaling has been described in a variety of human to LEF/TCF converts these proteins into transcriptional activators

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by displacing histone deacetylases (HDACs) and inhibitors of the elevation of inositol 1,4,5-trisphosphate (IP3) thereby releasing Groucho protein family followed by recruitment of the Legless Ca2+ from the endoplasmic reticulum (Kohn and Moon, 2005). family docking protein Bcl9, CBP/p300 and histone acetylases Increased cytosolic Ca2+ levels finally stimulate activity of pro- (Gordon and Nusse, 2006; Archbold et al., 2012). Activation of tein kinase C (PKC), calcium/calmodulin-dependent kinase II LEF-1/TCF then provokes transcription of numerous genes con- (CamKII) and calcineurin which provoke nuclear recruitment of trolling developmental processes, cell cycle, differentiation and nuclear factor κB (NFκB) and of nuclear factor of activated T cell invasion such as cyclin D1, c-myc, c-jun, MMPs, urokinase cells (NF-AT) (Saneyoshi et al., 2002; Ma and Wang, 2006; De, plasminogen activator receptor (uPAR), Notch signaling factors 2011). In addition, Ca2+ accumulation upon Wnt5a stimulation and many others depicted at the Wnt homepage1. Moreover, Wnt can induce TGF-β-activated kinase (TAK1) and Nemo-like kinase signaling components such as Axin, TCFs or Fzds are often con- (NEMO), which block TCF through phosphorylation and antag- trolled by a feedback loop upon Wnt activation. Accumulating onize canonical Wnt signaling (Ishitani et al., 1999; Ishitani and evidence, however, suggests that our view of canonical Wnt sig- Ishitani, 2013). Hence, in some cancers Wnt5a acts as a tumor naling is still too simplistic. Proteins of the destruction complex suppressor. However, the effects of Wnt5a strongly depend on the can enter the nucleus and influence trafficking of Wnt signaling receptor context. Binding to Fzd2, 3, 5, 6 induces Ca2+ signaling, components as well as gene transcription. Numerous soluble neg- but the ligand can also activate the canonical pathway upon inter- ative regulators of Wnt signaling such as different Dickkopf (Dkk) action with Fzd4 and LRP (Mikels and Nusse, 2006; Nishita et al., or secreted frizzled-related proteins (sFRPs) binding to Wnts 2010). Moreover, various studies suggest that different Wnts can and LRP, respectively, have been identified (Gordon and Nusse, interact with the receptor tyrosine kinases Ryk and Ror2 and pro- 2006). TCFs interact with several other regulatory transcription mote developmental processes independently of Fzd or activate factors, for example Smads, c-jun or Cdx proteins, likely deter- classical signaling pathways such as ERK or PI3K/AKT/mTOR mining specificity of binding to Wnt response elements (Archbold (Yun et al., 2005; Kim et al., 2007; Fradkin et al., 2010; Minami et al., 2012). Moreover, TCF/β-catenin transcriptional complexes et al., 2010). In conclusion, Wnts binding to canonical and diverse can also repress transcription and β-catenin can be recruited to non-canonical receptors form a highly complex signaling network other transcription factors than LEF/TCF, e.g., steroid hormone with a considerable overlap between the different pathways. receptors, in a Wnt-dependent manner (Beildeck et al., 2010). Several alterations in Wnt signaling components have been Wnt SIGNALING IN PLACENTA AND TROPHOBLAST detected in cancer cells provoking nuclear accumulation of β- As outlined above and discussed elsewhere, early placental devel- catenin and aberrant Wnt signaling (Camilli and Weeraratna, opment is associated with rapid generation of several trophoblast 2010). While mutations in Wnt ligands are rare, mutations in subtypes forming distinct functional villi in mice and men (Geor- the APC tumor suppressor gene have been identified in the major- giades et al., 2002; Red-Horse et al., 2004). Likewise, the maternal ity of sporadic colorectal cancers. Moreover, activating mutations uterus adapts to pregnancy by extensive tissue remodeling involv- in β-catenin inhibiting its GSK-3β-dependent degradation were ing differentiation of stromal cells, angiogenesis and immunolog- detected in colon, prostate and other malign tumors. In addition, ical alterations. These critical processes are initiated during the there is also compelling evidence that epigenetic changes of the secretory phase of the menstrual cycle and upon implantation and Wnt pathway contribute to tumorigenesis since downregulation early stages of placental development. Given the fact that Wnt sig- of sFRP gene transcription through promoter methylation has naling is important for organ development and tissue homeostasis, been observed in different epithelial cancers (Herr et al., 2012). it may not be surprising that the pathway also has major roles in uterus formation, growth and differentiation. Gene targeting in NON-CANONICAL Wnt SIGNALING mice revealed that β-catenin and different Wnts, such as Wnt4, The fact that different Wnts can exert effects on cells indepen- Wnt5a, or Wnt7a, are critical for uterine development (Miller and β dently of -catenin adds further complexity to the particular Sassoon, 1998; Vainio et al., 1999; Mericskay et al., 2004; Arango signaling pathway. Ligands such as Wnt5a or Wnt11 can acti- et al., 2005). Wnt signaling components also play a role in stroma 2+ vate the Wnt/PCP and the Wnt/Ca pathway by binding to Fzds cell proliferation and differentiation for example Wnt4, Wnt6 (Li and activating Dvl independently of LRP-5 or -6 (Komiya and et al., 2013; Wang et al., 2013) or Dkk1, a progesterone-regulated Habas, 2008). Induction of Wnt/PCP signaling, originally identi- gene which is induced in the endometrium upon decidualization fied in different developmental processes of Drosophila, involves (Tulac et al., 2006; Duncan et al., 2011). the G-proteins Rac and RhoA and the downstream effectors c-jun This review focuses on function of Wnt signaling in trophoblast NH2-terminal kinase (JNK) and Rho-associated kinase (ROCK), and placental development and differentiation, whereas a number respectively. Wnt/PCP signaling is critically involved in the forma- of different papers summarize the role of canonical and non- tion of embryonic tissues and organs and aberrant activation of canonical Wnt signaling in female reproductive tract development the pathway was shown to promote metastasis of different cancer and differentiation, uterine function and decidualization (Chen types (Wang, 2009). et al., 2009; Sonderegger et al., 2010b; van der Horst et al., 2012; 2+ On the other hand, Wnt/Ca signaling inhibits cGMP- Wetendorf and DeMayo, 2012). dependent protein kinase (PKG), which blocks Ca2+ release in unstimulated cells and activates phospholipase C (PLC) and Wnt SIGNALING IN MURINE PLACENTAL DEVELOPMENT Expression of several Wnt ligands, Fzds and Dvl proteins has 1http://www.stanford.edu/˜rnusse/wntwindow.html been noticed in the developing murine blastocyst during the

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pre-implantation period (Mohamed et al., 2004; Harwood et al., a positive feedback loop of Fzd5 and GCM1 controls different steps 2008). However, the canonical pathway does not seem to play a role of placental morphogenesis promoting branchpoint initiation, in blastocyst formation. Embryos bearing homozygous deletion of chorionic trophoblast-specific vEGF expression and trophoblast β-catenin develop to the blastocyst stage but are affected upon gas- syncytialisation (Lu et al., 2013). Placental phenotypes of mice trulation (Haegel et al., 1995). However, maternal β-catenin may with homozygous deletions of Wnt signaling genes are summa- have compensated the lack of embryonic β-catenin. Therefore, rized in Table 1. Early stages of murine trophoblast invasion, mothers harboring a conditional deletion of the gene in oocytes however, might be negatively affected by canonical Wnt signal- were additionally used in β-catenin knock-out studies (De Vries ing since recombinant Dkk1 was shown to increase motility in et al., 2004). Again, development into blastocysts was observed co-cultivations of ectoplacental cones with decidual cells (Peng suggesting that β-catenin is not required for early pre-implantation et al., 2008). development. Also, treatment with Dkk1 did not impair blasto- cyst formation (Xie et al., 2008). Non-canonical signaling such as EXPRESSION PATTERNS OF Wnt LIGANDS AND FRIZZLED RECEPTORS 2+ the Wnt/Ca pathway could be involved (Chen et al., 2009). On IN HUMAN TROPHOBLASTS the other hand, there is evidence that the canonical Wnt pathway Human trophoblast development is mostly studied in choriocar- could regulate blastocyst development in other species for example cinoma model systems and immortalized trophoblast cell lines, in ruminants (Denicol et al., 2013). since primary placental material can only be obtained from very Whereas the canonical Wnt pathway is dispensable for murine restricted time points during pregnancy. Trophoblast cell fusion blastocyst development, it is critical for blastocyst activation, can be investigated in isolated term CTBs involving numerous adhesion and implantation. Inhibition of the pathway through effectors (Morrish et al., 1998). Invasive properties of cells are lost Dkk1 or small molecular inhibitors decreased implantation, which at the end of pregnancy and therefore trophoblast motility and was also shown to be associated with induction of canonical differentiation of progenitors into EVTs has to be analyzed in first β-catenin and downregulation of non-canonical Wnt-RhoA sig- trimester placentae using villous explant cultures and/or isolated naling (Xie et al., 2008). Likewise, treatment with sFRP2 was CTBs (Pollheimer and Knöfler, 2005). Investigations in these pri- shown to decrease implantation rates (Mohamed et al., 2005). mary cultures as well as in cell lines provided evidence for an Induction of the canonical pathway in the uterine epithelium autocrine role of Wnt signaling in human trophoblast prolifera- and myometrial smooth muscle cells at the site of implanta- tion and invasion (Sonderegger et al., 2010b).Asafirststep,our tion through trophectoderm-derived Wnt ligands seems to be laboratory analyzed the expression patterns of all Wnt ligands and important (Mohamed et al., 2005). Fzd receptors in different trophoblast cell lines, isolated CTBs and Wnt signaling could also play a key role during early trophoblast development in mice. Treatment of embryonic stem cells with Wnt3a induced formation of trophectodermal stem cells with the capacity to differentiate into spongiotrophoblasts and giant Table 1 | Placental phenotypes of mice with homozygous deletion of Wnt signaling components. cells which was mediated through LEF-1-dependent induction of

Cdx2, a key regulator of early trophoblast lineage determination Gene Phenotype Reference (Chawengsaksophak et al., 2004; He et al., 2008). knock-out Furthermore, gene targeting in mice provided evidence that Wnt signaling components control sequential steps of placen- Wnt2 Defects in labyrinthine zone Monkley et al. tal development. In particular, chorioallantoic fusion, branching Decreased number of fetal capillaries (1996) morphogenesis, labyrinth development and placental angiogene- sis were affected in these mutants (Cross et al., 2006). For example, Oedema formation gene knock-outs of Wnt7b, R-spondin3, a soluble activator of Fibrinoid deposition canonical Wnt signaling, or of both TCF-1 and LEF-1 show defects Wnt7b Defect in alpha4-mediated Parr et al. (2001) in chorioallantoic fusion thereby altering normal labyrinth devel- chorioallantoic fusion opment and function at later stages of embryogenesis (Galceran Disorganization of chorionic plate et al., 1999; Parr et al., 2001; Aoki et al., 2007). Similar to the R-spondin3 Defects in chorioallantoic fusion, Aoki et al. (2007) above mentioned mutants, homozygous deletion of Wnt2 and Fzd5 affected branching and labyrinth formation, however, with- branching and labyrinth formation out alterations in chorioallantoic fusion (Monkley et al., 1996; Reduced expression of Gcm1 Ishikawa et al.,2001). Moreover, placentae lacking Bcl-9, one of the Fzd5 Defects in yolk sac angiogenesis and Ishikawa et al. co-activators of LEF-1/TCF, showed defective branchpoint initia- placental vasculogenesis (2001); Lu et al. tion and a decrease in syncytiotrophoblast formation (Matsuura Defective chorioallantoic branching (2013) et al.,2011). GCM1, the key transcription factor in branching mor- Decreased GCM1 expression phogenesis and trophoblast cell fusion (Anson-Cartwright et al., Bcl9 Defective branching initiation Matsuura et al. 2000), is probably the most critical Wnt target in mouse pla- cental development since diminished expression of GCM1 was Failures in trophoblast syncytialisation (2011) noticed in R-spondin3 and Bcl-9-mutant placentae (Aoki et al., LEF-1/TCF-1 Defects in chorioallantoic fusion Galceran et al. 2007; Matsuura et al., 2011). Indeed, a recent study indicated that (1999)

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total placental extracts of first and third trimester, as well as in villi to induce TCF/β-catenin-dependent transcription but antago- and EVTs obtained from first trimester villous explant cultures nized the canonical pathway in trophoblasts (Sonderegger et al., using optimized, semi-quantitative RT-PCR (Sonderegger et al., 2007). Progesterone treatment of ovariectomized mice was shown 2007). 14 out of 19 Wnt ligands, and 8 out of 10 Fzd receptors were to stimulate Wnt11 expression, another non-canonical Wnt lig- found to be expressed in total first trimester placenta. Most of these and, whereas Wnt4 and Wnt7b are induced by estrogen which mRNAs were detectable in the villous trophoblast epithelium. In likely has implications for implantation (Hayashi et al., 2009) and particular, abundantly (Wnt1, Wnt2b, Wnt4, Wnt7b, Wnt10a, decidualization as mentioned above. Interestingly, Wnt4, Wnt11 Wnt10b, Wnt11), moderately (Wnt5a, Wnt9b) and lowly (Wnt2, and Wnt7b are among the most abundantly produced Wnts in Wnt3, Wnt5b, Wnt6, Wnt7a) -expressed Wnt ligands were present CTBs and Wnt7b expression decreased during EVT formation in first trimester CTBs as depicted (Figure 2). Hence, expression of (Figure 2). Considering that Wnt7b is required for mouse placen- canonical (for example Wnt1, Wnt2b, Wnt7b, Wnt10a, Wnt10b) tal development (Parr et al., 2001), the human homologue may as well as non-canonical Wnts (Wnt4, Wnt5a, Wnt11) suggests also play a role in placenta formation and/or control critical func- that different Wnt pathways may operate during human tro- tions during the first trimester of pregnancy such as trophoblast phoblast development. Wnt1, Wnt7b, Wnt10a, and Wnt10b were proliferation. Similarly, Wnt10a, Wnt10b, Fzd5, and Fzd10 were downregulated from first trimester to term suggesting roles in tro- strongly expressed in CTBs but largely absent from EVTsor placen- phoblasts of early pregnancy (Sonderegger et al., 2007). Whereas tal fibroblasts suggesting that these Wnt and Fzd members are also Fzd2 and Fzd4 are only produced in villous mesenchymal cells, predominantly associated with early trophoblast cell growth. The Fzd1, Fzd3, Fzd5, Fzd6, Fzd7, and Fzd10 are expressed in CTBs. principal Fzd receptor in EVTs is Fzd6, which operates through The latter was absent from villous fibroblasts suggesting a specific canonical as well as non-canonical pathways (Golan et al., 2004; role in CTBs. Although none of the individual Wnts and Fzds Wu et al., 2009). Differential expression of Fzds between CTBs and have been studied in the context of human trophoblast invasion EVTs is also detectable in our previously established gene expres- and differentiation so far, expression patterns and comparison to sion profiles (Bilban et al., 2009) as shown in Figure 3. Whereas the the situation in mouse placenta and other Wnt-dependent systems role of Wnt10a, Wnt10b or FZD10 in murine placental develop- in reproduction allows to speculate about their potential roles. ment is unknown, Fzd5 is critical for branching morphogenesis Wnt4 is produced by CTBs as well as EVTs and could contribute as mentioned before (Lu et al., 2013). Hence, Fzd5 could also to decidualization through the canonical pathway (Li et al., 2013). play a crucial role in human placental development. Furthermore, Wnt5a, secreted from trophoblast cell lines and primary cultures, another semi-quantitative RT-PCR study suggested that several of may act through non-canonical pathways since it was unable the above mentioned Wnt ligands are regulated in a gestation- dependent manner in first trimester placenta (Grisaru-Granovsky et al., 2009) suggesting that specific combinations of Wnts could eventually control sequential steps of human trophoblast func- tion and/or development. Moreover, the endometrial Wnt ligands Wnt2, Wnt4, Wnt5a, Wnt7a, Wnt8b, and Wnt3, the latter being regulated during the menstrual cycle, could affect trophoblast function in a paracrine manner (Tulac et al., 2003).

Wnt SIGNALING IN HUMAN TROPHOBLAST FUNCTION AND DIFFERENTIATION Studies in mice suggested that Wnt signaling is critical for acti- vation and implantation of blastocysts (Mohamed et al., 2005). In vitro investigations with trophoblast cell lines and cultures also revealed a role of the particular signaling pathway in human trophoblast adhesion, invasion and differentiation. Treatment of decidualized stromal cells with supernatants of trophoblasts provoked changes in the expression of some Wnt signaling components suggesting that soluble trophoblast-derived factors could influence endometrial function and differentiation through regulation of the Wnt pathway (Hess et al., 2007). Differ- entWnts secreted from EVTscould be the prime factors controlling expression of decidual Wnt signaling components since many of these genes are direct targets of TCF/β-catenin as summarized at the Wnt homepage1. Along those lines, attachment of spheroids FIGURE 2 | Schematic illustration of transcript levels encoding Wnt prepared from JAR choriocarcinoma cells to endometrial Ishikawa ligands and Fzd receptors in isolated human first trimester CTBs and cells was inhibited in the presence of Dkk1 (Liu et al., 2010). EVTs measured by semi-quantitative RT-PCR as published elsewhere Similarly, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a nega- (Sonderegger et al., 2007). Low, medium and high levels of expression are indicated by one, two and three black squares, respectively. tive effector of implantation, decreased attachment of JEG-3 or BeWo cell spheroids to different endometrial epithelial cell lines

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level (Figure 3). Moreover, stimulation with a Wnt ligand increased invasion of primary CTBs and trophoblastic SGHPL- 5 cells in transwell assays and promoted migration in villous explant cultures seeded on collagen I, which was inhibited upon treatment with recombinant Dkk1 (Pollheimer et al., 2006; Son- deregger et al., 2010a). In addition, basal migration and invasion of the different trophoblast models were reduced in the pres- ence of Dkk1 suggesting that the aforementioned canonical Wnts expressed in EVTs exert autocrine effects. Wnt stimulation was also shown to activate non-canonical AKT signaling and AKT- dependent motility of trophoblasts (Sonderegger et al., 2010a). Canonical LRP-5/6-FZD receptors were not involved since Wnt- dependent phosphorylation of AKT could not be inhibited upon supplementation of Dkk1. Also, cross-talk between the canonical Wnt pathway and AKT through AKT-induced phosphorylation and inactivation of GSK-3β as mentioned for other cells (Naito et al., 2005) has not been observed in trophoblasts since chemical AKT inhibitors did neither change nuclear accumulation of β- catenin nor the activity of a canonical Wnt reporter (Sonderegger et al., 2010a). One of the putative targets increasing invasive- ness in a Wnt-dependent manner could be MMP-2, which is elevated in trophoblast supernatants upon Wnt stimulation and also has been described as a direct target of TCF/β-catenin (Wu et al., 2007; Sonderegger et al., 2010a). Although MMP-2 mRNA expression is associated with EVT differentiation (Figure 3), tran- script levels did not change upon Wnt stimulation suggesting that the pathway induces MMP-2 secretion or affects its stability (Sonderegger et al., 2010a). Although canonical Wnt signaling is strongly elevated upon EVT formation, CTBs also respond to Wnt signals. Wnt- dependent activation of TCF/β-catenin provoked increased prolif- eration and expression of the cell cycle regulator cyclin D1 as well as induction of Wnt signaling components (Pollheimer et al., 2006). Although TCF-4 is absent from proliferative CTBs, the canonical pathway might be activated through TCF-3 which is present in both CTBs and EVTs (Figure 3). Furthermore, epigenetic analyses provided evidence for a gen- eral activation of Wnt signaling in placental tissues and isolated trophoblasts. Genes encoding negative regulators of the pathway, FIGURE 3 | Color-coded mRNA expression (GDS3523) in non-invasive i.e., APC, sFRP2 and engrailed-1 were shown to be hypermethy- CTBs (5 pools) and invasive EVTs (6 pools) analyzed by GEO DataSet lated in trophoblasts, whereas these changes were not observed in Cluster Analysis online tool (http://www.ncbi.nlm.nih.gov/geo/). Markers of EVT differentiation, Fzd receptors, putative TCF-4/β-catenin placental fibroblast or leukocytes (Novakovic et al., 2008; Wong target genes as well as biomarkers of EMT are depicted. et al., 2008). This suggests that specific activation of the path- way in trophoblasts could play a role in placentation. In addition, other effectors than Wnt ligands likely contribute to stabilization by suppressing β-catenin, which could be reverted upon treatment of β-catenin and TCF/β-catenin-dependent trophoblast prolifer- with a recombinant Wnt ligand (Tsang et al., 2012). ation and invasion. Activation of protease activated receptor-1 Moreover, expression analyses of TCFs in human first trimester (PAR1) provoked an increase in these processes whereas siRNA- placenta suggested that the canonical Wnt pathway is associ- mediated gene silencing of PAR1 or addition of soluble inhibitors ated with the invasive differentiation process of trophoblasts downregulated TCF/β-catenin-induced proliferation and motility (Pollheimer et al., 2006). Immunofluorescence of tissues revealed (Grisaru-Granovsky et al., 2009). Expression of StarD7, a member induction of TCF-4 protein in the nucleus of non-proliferating, of the StAR1 lipid transfer proteins promoting proliferation and p57/KIP2-positive EVTs as well as nuclear recruitment of β- invasion of choriocarcinoma cells, was shown to be directly con- catenin in a considerable number of these cells. Analyses of our trolled by TCF/β-catenin (Rena et al., 2009; Flores-Martin et al., expression profiles performed with total mRNA isolated from five 2012). and six different CTB and EVT cell preparations, respectively In summary, the present literature implicates canonical Wnt (Bilban et al., 2009), suggested induction of TCF-4 at the mRNA signaling in human trophoblast proliferation and invasion.

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However, the question of whether TCF molecules are indeed recruitment of β-catenin and increased expression of TCF-4 in regulators of EVT formation, in other words control the switch EVTs drives a set of genes promoting trophoblast invasion and from proliferation to cell cycle arrest and differentiation is still migration. unknown and currently under investigation in our laboratory. Another molecular process critically involved in cancer cell So far, canonical Wnt signaling has been identified as a regu- invasion and metastasis is epithelial to mesenchymal transi- lator of trophoblast cell fusion. GCM1, the most critical factor tion (EMT), in which epithelial cells lose their polarity and in syncytialisation controlling expression of the fusogenic pro- gain fibroblast-like properties promoting invasion and migration teins syncytin-1 and -2, harbors TCF binding sites in one of its (Zheng and Kang, 2013). Interestingly, EMT in cancer cells also introns and silencing of TCF-4 or β-catenin impaired cAMP- provokes growth arrest and cells have to revert back to an epithelial induced cell fusion of BeWo choriocarcinoma cells (Matsuura phenotype (MET) allowing for cell growth and distant metastasis et al., 2011). Moreover, Wnt targets such as Axin, BMP and activin formation (Brabletz, 2012). Analyses of gene expression profiles membrane-bound inhibitor (BAMBI), and LEF-1 increased upon suggest that EMT also occurs in invasive trophoblasts (Figure 3) elevation of cAMP. However, it has to be mentioned that LEF-1 which have stopped proliferation allowing for differentiation to is only expressed in stromal cells of human placentae and TCF-3 take place. Although invasive trophoblasts do not induce mes- and -4 are absent from villous CTBs after the 6th week of ges- enchymal vimentin, at least in vitro, and maintain expression tation (Pollheimer et al., 2006). Since trophoblast fusion occurs of the epithelial marker cytokeratin 7, they upregulate typical until the end of pregnancy, Wnt-dependent GCM1 expression EMT-associated mRNAs such as heat shock protein 47 (HSP47), and syncytialization may only operate during very early stages Snail, ITGA5, ITGAB1, fibroblast-specific protein 1 (FSP1), MMP- of human gestation. Similar to mice, Wnt signaling might also 2, and secreted protein acidic and rich in cysteine (SPARC) play a role in early human trophoblast lineage determination and downregulate genes associated with cell-cell adhesion and since the pathway was found to be activated upon BMP4- epithelial polarity such as the tight junction proteins occludin mediated differentiation of embryonic stem cells into trophoblasts (OCLN) and ZO1. Also, transient loss of adherens junction pro- (Marchand et al., 2011). teins, i.e., membrane-bound β-catenin and E-cadherin, has been detected in the proximal invasion zone of anchoring villi using Wnt TARGET GENES AND THE ROLE OF Wnt-DEPENDENT immunofluorescence in first trimester placental tissues (Zhou TRANSCRIPTION FACTORS IN EPITHELIAL TO MESENCHYMAL et al., 1997b). Elevated Wnt signaling, expression of LEF/TCF TRANSITION and nuclear recruitment of β-catenin is also a typical feature of Numerous studies indicated that cancer cell invasion shares several EMT (Moustakas and Heldin, 2007). Hence, similar to cancer features with trophoblast invasion although the latter is precisely cells, induction of TCF-4 upon invasive trophoblast differentiation controlled in time and space. Besides expression of proteases, EVTs could orchestrate an EMT-like program to promote cell motility. produce critical such as the fibronectin receptor integrin In this process, TCF-4 could act as master regulator since it may α5β1 and the collagen/laminin receptor integrin α1β1 promoting not only directly control expression of pro-migratory EMT genes trophoblast adhesion and migration (Damsky et al., 1994; Aplin but also activate other critical key regulatory transcription factors et al., 1999). Conversely, EGF receptor 1 (EGFR1), indicative for inducing EMT such as Snail, Slug or ZEB1 (Medici et al., 2008; the proliferative capacity of trophoblasts as well as integrin α6 Sanchez-Tillo et al., 2011). (ITGA6), a marker of the polarized epithelium, were downreg- ulated during EVT differentiation (Jokhi et al., 1994). Changes Wnt signaling in gestational diseases in the mRNA expression pattern of genes involved in EVT inva- Changes in gene expression as well as in epigenetic modifica- sion and differentiation can also be monitored in our published tions of Wnt signaling components were shown to be associated chip data (Bilban et al., 2009) which are accessible via GEO pro- with different gestational diseases. Compared to normal tissues, files2. ITGA6 and EGFR are highly expressed in the five different higher numbers of β-catenin-positive EVT nuclei were detected CTB cell pools but weakly present in the six EVT preparations in placentae of complete hydatidiform mole (CHM) suggesting confirming the published literature (Figure 3). In contrast, HLA- that aberrant Wnt signaling could contribute to abnormal inva- G1, and the pro-migratory genes integrin α1 (ITGA1), integrin α5 sion in this pregnancy disorder (Pollheimer et al., 2006). Genes (ITGA5) and fibronectin 1 (FN1) were upregulated in EVTs. Inter- encoding APC and sFRP2 were shown to be hypermethylated in estingly, various mRNAs such as BAMBI, a marker of metastasis choriocarcinoma cells, indicating that inactivation of negative in colon cancer (Fritzmann et al., 2009), insulin-like growth fac- regulators of Wnt signaling likely contributes to the forma- tor 2 (IGF2), inducible nitric oxide synthase (iNOS), fibronectin 1 tion and/or progression of trophoblastic cancer cells (Novakovic (FN1), MMP-2, Notch2, uPAR, and Snai1 (also known as Snail1), et al., 2008; Wong et al., 2008). Similarly, Dkk1 was found to which are all direct targets of TCF/β-catenin1, were found to be absent from choriocarcinoma cells and re-expression of the be increased in the EVT pools concomitant with the upregu- gene induced growth arrest and apoptosis suggesting that the lation of TCF-4 (Figure 3). Indeed, these genes have already loss of Dkk1 is critical for tumor cell proliferation (Peng et al., been implicated in the control of trophoblast motility (Bischof 2006). In contrast, Dkk1 and sFRP4 were increased whereas Wnt2 et al., 2000; Lala and Chakraborty, 2003; Harris et al., 2008; and β-catenin were decreased in tissues of preeclamptic patients Hunkapiller et al., 2011). Therefore, we speculate that nuclear (Zhang et al., 2013a,b). Therefore, downregulation of the pathway could contribute to failed placentation and shallow trophoblast 2http://www.ncbi.nlm.nih.gov/geoprofiles/?term=GDS3523[ACCN] invasion observed in these pregnancies. Along those lines, elevated

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CONCLUSIONS In conclusion, the present literature suggests that Wnt signaling is critical for physiological processes of human trophoblasts. The pathway could play a role in blastocyst adhesion, implantation and early trophoblast lineage decisions. Moreover, Wnt signaling could regulate trophoblast cell fusion as well as development of the anchoring villus controlling proliferation, invasion and dif- ferentiation. In first trimester placenta numerous canonical and non-canonical Wnt ligands are expressed in CTBs and likely regu- late proliferation or other functions of these cells through the most abundant receptors, i.e., Fzd5 and Fzd10 (Figure 4). Differentia- tion of CTBs into growth-arrested invasive EVTs is associated with increased expression of TCF-4, nuclear recruitment of β-catenin and elevated canonical Wnt activity controlling trophoblast motil- ity potentially through Fzd6. Furthermore, TCF-4/β-catenin could activate a set of genes promoting invasion as well as the EMT-like features of migratory trophoblasts. Hence, Wnt signaling likely contributes to the strong, intrinsic differentiation program of EVTs and their inherently invasive properties. Hyperactivation of autocrine Wnt signaling could play a role in trophoblast dis- orders with elevated proliferation and invasion such as CHM and choriocarcinomas whereas downregulation of the pathway could be a cause of impaired placentation and trophoblast invasion observed in preeclampsia. Epigenetic changes such as methyla- FIGURE 4 | Model system for the role of Wnt signaling in function and differentiation of the human anchoring villus. Wnt ligands and Fzd tion of negative regulators of Wnt signaling could contribute to receptors expressed in vCTBs/CCTs and EVTs are shown. During EVT the induction of Wnt signaling likely promoting normal placen- formation, Wnt7b, Wnt9b, Wnt10a, Wnt10b, Fzd5, and Fzd10 are down- tation as well as trophoblast tumor progression. Besides autocrine regulated suggesting a role in trophoblast proliferation. However, interstitial control decidual Wnt ligands could modulate trophoblast func- cytotrophoblasts (iCTBs) upregulate TCF-4 and nuclear β-catenin to promote trophoblast motility and possibly EVT differentiation. tion via canonical and/or non-canonical pathways. The fact that Dkk1 is expressed in the decidua and increased upon progesterone treatment could suggest a mechanism to restrain the extent of levels of Dkk1 were detected in women with recurrent abortions trophoblast invasion. Further studies are needed to delineate spe- (Bao et al., 2013). Interestingly, cytomegalovirus (CMV) infection, cific Wnt-Fzd interactions in human trophoblasts and to define a putative cause of spontaneous abortion and preterm delivery, the role of non-canonical Wnt signaling in normal and aberrant was shown to decrease trophoblast proliferation and invasion trophoblast proliferation, invasion and differentiation. involving inhibition of Wnt signaling. Infection of trophoblastic SGHPL-4 cells with the virus altered the localization of β-catenin ACKNOWLEDGMENTS and induced its degradation as well as downregulation of a canon- Research in the laboratory is supported by grants of the Austrian ical Wnt reporter (Angelova et al., 2012). Some of these effects Science Fund (P-22687-B13 to Martin Knöfler and P-25187- might be exerted via peroxisome proliferator-activated receptor γ B13 to JJürgen Pollheimer) as well as by grant AP00574OFF of (PPAR- γ) a negative regulator of trophoblast invasion, which is the Herzfelder´sche Familienstiftung and by grant 14147 of the activated upon CMV infection and was shown to provoke pro- Austrian National Bank donated to Martin Knöfler and Jürgen teasomal degradation of β-catenin in other cells (Liu et al., 2006; Pollheimer, respectively. We thank V. Fock for critical reading of Rauwel et al., 2010). the manuscript.

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Methylation Zhang, Z., Zhang, L., Jia, L., Wang, mercial or financial relationships that 10.1242/dev.033910 of the adenomatous polyposis coli P., and Gao, Y. (2013b). Associ- could be construed as a potential con- van der Horst, P. H., Wang, Y., Van Der (APC) gene in human placenta ation of Wnt2 and sFRP4 expres- flict of interest. Zee, M., Burger, C. W., and Blok, and hypermethylation in chorio- sion in the third trimester placenta L. J. (2012). Interaction between sex carcinoma cells. Cancer Lett. 268, in women with severe preeclamp- Received: 26 June 2013; accepted: 06 hormones and WNT/beta-catenin 56–62. doi: 10.1016/j.canlet.2008.03. sia. Reprod. Sci. 20, 981–989. doi: September 2013; published online: 26 signal transduction in endometrial 033 10.1177/1933719112472740 September 2013. physiology and disease. Mol. Cell. Wu, B., Crampton, S. P., and Hughes, Zheng, H., and Kang, Y. (2013). Citation: Knöfler M and Pollheimer Endocrinol. 358, 176–184. doi: C. C. (2007). 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Dysregulation of Friz- is associated with failure of human Frontiers in Genetics. 521826. doi: 10.1155/2011/521826 zled 6 is a critical component of B-cell cytotrophoblasts to mimic a vascu- Copyright © 2013 Knöfler and Poll- Wang, Q., Lu, J., Zhang, S., Wang, S., leukemogenesis in a mouse model of lar adhesion phenotype. One cause heimer. This is an open-access article dis- Wang, W., Wang, B., et al. (2013). chronic lymphocytic leukemia. Blood of defective endovascular invasion tributed under the terms of the Creative Wnt6 is essential for stromal cell 113, 3031–3039. doi: 10.1182/blood- in this syndrome? J. Clin. Invest. Commons Attribution License (CC BY). proliferation during decidualization 2008-06-163303 99, 2152–2164. doi: 10.1172/JCI11 The use, distribution or reproduction in in mice. Biol. Reprod. 88, 5. doi: Xie, H., Tranguch, S., Jia, X., Zhang, 9388 other forums is permitted, provided the 10.1095/biolreprod.112.104687 H., Das, S. K., Dey, S. K., et al. Zhou, Y., Fisher, S. J., Janatpour, M., original author(s) or licensor are cred- Wang, Y. (2009). Wnt/planar cell polar- (2008). Inactivation of nuclear Wnt- Genbacev, O., Dejana, E., Wheelock, ited and that the original publication in ity signaling: a new paradigm for beta-catenin signaling limits blasto- M., et al. (1997b). Human cytotro- this journal is cited, in accordance with cancer therapy. Mol. Cancer Ther. cyst competency for implantation. phoblasts adopt a vascular phenotype accepted academic practice. No use, dis- 8, 2103–2109. doi: 10.1158/1535- Development 135, 717–727. doi: as they differentiate. A strategy for tribution or reproduction is permitted 7163.MCT-09-0282 10.1242/dev.015339 successful endovascular invasion? J. which does not comply with these terms.

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