Signalling pathways regulating the invasive differentiation of human trophoblasts: a review

Placenta (2005), Vol. 26, Supplement A, Trophoblast Research, Vol. 19 doi:10.1016/j.placenta.2004.11.013

Signalling Pathways Regulating the Invasive Differentiation of Human Trophoblasts: A Review J. Pollheimer and M. Kno¨fler* Department of Obstetrics and Gynecology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria Paper accepted 23 November 2004

The invasive differentiation pathway of trophoblasts is an indispensable physiological process of early human placental development. Formation of anchoring villi, proliferation of cell columns and invasion of extravillous cytotrophoblasts into maternal decidual stroma and vessels induce vascular changes ensuring an adequate blood supply to the growing fetus. Extravillous trophoblast differentiation is regulated by numerous growth factors as well as by extracellular matrix proteins and adhesion molecules expressed at the fetalematernal interface. These regulatory molecules control cell invasion by modulating activities of matrix-degrading protease systems and ECM adhesion. The differentiation process involves numerous signalling cascades/proteins such as the GTPases RhoA, the protein kinases ROCK, ERK1, ERK2, FAK, PI3K, Akt/protein kinase B and mTOR as well as TGF-b-dependent SMAD factors. While an increasing number of signalling pathways regulating trophoblast differentiation are being unravelled, downstream effectors such as executing transcription factors remain largely elusive. Here, we summarise our current knowledge on signal transduction cascades regulating invasive trophoblast differentiation. We will focus on cell model systems which are used to study the particular differentiation process and discuss signalling pathways which regulate trophoblast proliferation and motility. Placenta (2005), Vol. 26, Supplement A, Trophoblast Research, Vol. 19 Ó 2005 Published by IFPA and Elsevier Ltd. Keywords: Human trophoblast; Signal transduction; Differentiation; Cell invasion Abbreviations: ALKs, activin-receptor-like kinases; bHLH, basic helix-loop-helix; CaMKII, Ca/calmodulin-dependent kinase II; CTB, cytotrophoblast; eCTB, endovascular cytotrophoblast; ECM, extracellular matrix; EGF, epidermal growth factor; EMSA, elotrophorectic mobility shift assay; ERK, extracellular signal-regulated kinase; EVT, extravillous trophoblast; FGF, fibroblast growth factor; FAK, focal adhesion kinase; GAP, GTPase activating protein; GEF, guanine nucleotide exchange factor; GSK-3b, glycogen synthase kinase 3b; GPCR, G-protein-coupled receptor; hCG, human chorionic gonadotrophin; HGF, hepatocyte growth factor; ICTB, interstitial cytotrophoblast; IGF, insulin-like growth factor; IGFBP, insulin-like growth factor binding protein; IkB, inhibitor of kB; JAK, Janus kinase; JNK, c-Jun N-terminal kinase; LRP-5, low density lipoprotein receptor-related protein 5; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; mTOR, mammalian target of rapamycin; PAK, p21-activated kinase; PI3K, phosphoinositide 3-kinase; PIP2, phosphatidylinositol-4,5bis-phosphate; PKC, protein kinase C; PAI-1, plasminogen activator inhibitor 1; PTEN, phosphatase and tensin homolog deleted on chromosome 10; PTK, protein tyrosine kinase; ROCK, Rho-associated-kinase; RTK, receptor tyrosine kinase; STAT, signal transducer and activator of transcription; S, syncytium; TCP, T-cell factor; TGF-b, transforming growth factor-b; uPA, urokinase plasminogen activator; vEGF, vascular endothelial growth factor; Wnt, vertebrate homologue of wingless.

INTRODUCTION Cellular signalling is controlled by protein kinases via sequential steps of protein phosphorylation representing the most common controlling mechanism of protein function in * Corresponding author. Tel.: C43 1 40400 2842; fax: C43 1 40400 7842. E-mail address: [email protected] (M. Kno¨fler). 0143e4004/$esee front matter

the cell. Signalling cascades are initiated by various stimuli such as growth factors, cytokines, hormones, extracellular matrix adhesion and cell to cell contact, governing multiple cellular responses. Hence, at the cell membrane integrin receptors, receptor tyrosine kinases (RTKs) and G-proteincoupled receptors (GPCRs) transduce extracellular signals that activate various cascades such as focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK) or the PI3K (phosphoinositide 3-kinases)/Akt pathway. All of these Ó 2005 Published by IFPA and Elsevier Ltd.

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cascades have been implicated in the control of a diverse range of biological processes, including cell cycle control, differentiation, cell migration and apoptosis [1e5]. Signalling became a main topic in molecular biology over the past years, since deregulation is linked with diseases such as cancer. Consequently, it is of great interest to study signalling pathways in the context of their specific role in pathological but also in physiological processes. Extravillous trophoblast formation represents a remarkable example of a physiological differentiation process, which is controlled by fetal as well as maternal factors of the placental bed [6,7]. Extravillous trophoblasts (EVT) originate from anchoring villi attached to the uterine stroma during the first weeks of pregnancy (Figure 1). Cytotrophoblast (CTB) stem cells residing at the villous basement membrane lose their polarised structure and form proliferating cell columns. At distal sites invasive EVT are generated which detach from the columns and invade decidual stromal compartments (interstitial CTB) as well as spiral arteries (endovascular cytotrophoblasts) of the decidua and upper part of the myometrium [8]. Differently to tumour cells, EVT quit cell growth and become polyploid (4Ne8N) during invasive differentiation [9]. In addition, endovascular trophoblasts invade maternal spiral arteries, replace the maternal endothelium and acquire a vascular adhesion phenotype [10]. These modifications contribute to the transformation of spiral arteries into vessels with low resistance thereby promoting uteroplacental circulation [11]. The lack of endovascular trophoblasts was noticed in the placental bed of women with preeclampsia or cases of severe intrauterine growth restriction (IUGR) [12e15] suggesting that failures in EVT formation/invasion could play a critical role. Therefore, elucidation of the regulatory mechanisms controlling differentiation of EVT in humans would be helpful to better understand the pathogenesis of the gestational diseases. Regarding signal transduction in trophoblast invasion various differential stages including proliferation, differentiation and migration/invasion need to be precisely coordinated and integrated at all times in order to guarantee successful placental development. Here, we review experimental data demonstrating a role of different signalling proteins such ERKs, PI3K, Rho/ROCK, FAK or PI3K/Akt/mTOR in extravillous trophoblast differentiation and discuss growth factors of the fetalematernal interface acting through these pathways. MODEL SYSTEMS FOR STUDIES OF INVASIVE TROPHOBLAST DIFFERENTIATION Various model systems have been developed to study human EVT differentiation/invasion in vitro. The most common way to study these processes is the use of tumorigenic (choriocarcinoma) or non-tumorigenic cell lines because they can be indefinitely propagated in the laboratory. Similarities/differences between established trophoblast cell lines with respect to trophoblast-specific gene expression have been investigated recently [16]. Here, the cell lines mainly used by investigators

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Figure 1. Formation and differentiation of extravillous trophoblasts. After anchorage of a mesenchymal villus at the uterine basement membrane cytotrophoblast (CTB) stem cells give rise to proliferative cell columns (CC). At distal sites non-proliferating, extravillous trophoblasts are formed which detach from the cell columns and migrate into stromal areas of the maternal decidua (formation of interstitial cytotrophoblasts, ICTB). ICTB differentiate to giant cells (GC) in deeper areas of the placental bed. Endovascular trophoblasts (ECTB), which play a role in remodelling of the vasculature, migrate into spiral arteries, replace the maternal endothelium (EC) and acquire molecular characteristics of endothelial cells. In floating villi surrounded by maternal blood, CTB stem cells fuse to form the multinucleated syncytium (S).

are described. HTR-8 cells and its SV40 large T antigentransformed derivative, HTR-8/SVneo cells, have been generated from adherent cells after plating of minced chorionic villi of first trimester placental tissue [17,18]. Cells generated this way express markers of the EVT such as a1, a3, a5 and b1 integrin subunits [19]. The two cell lines share characteristics of EVT such as expression of cytokeratin 8 and 18 and

Pollheimer and Kno¨fler: Signalling Pathways Regulating Invasive Trophoblast Differentiation

production of the preform of MMP-2 [18]. Similarly, SGHPL-4 and SGHPL-5 cells have been produced after trypsinisation, gradient centrifugation and SV40 large T antigen transfection of minced first trimester placentae [20,21]. Expressions of several presumptive EVT proteins such as cytokeratin 18 and a1, a3, b1 integrins were noticed in SGHPL-5 cells [20]. SGHPL-4 cells migrate into fibrinembedded spiral arteries in vitro suggesting that the cell line may have retained cellular functions of the in vivo EVT [22]. However, studies using virus-transformed cells should also be interpreted in the context of artefacts introduced during selection and establishment of the cell line. Divergences between cell lines and primary EVTs may lead to difficulties in extrapolating in vitro effects to the in vivo situation. For example, trophoblastic cell lines continuously proliferate under different experimental settings whereas EVTs in situ and in vitro are differentiated, non-proliferating cells. Considering these aspects, additional trophoblast model systems have been established. Primary CTBs are isolated from first trimester villous material by trypsin treatment, Percoll gradient centrifugation and immunopurification [23,24]. Pure preparations are a mixture of hCG-expressing villous trophoblasts and EVTs [25], but cultures switch to an invasive phenotype upon plating on Matrigel. Extended experimental studies are hampered by the fact that isolated cell numbers are low and often contaminated with varying amounts of villous stromal cells. The villous explant culture system allows investigating EVT differentiation in a time- and distance-dependent manner without previous disruption of the villous structure. Mesenchymal villi are dissected from first trimester villi and seeded on ECM-coated dishes. In vitro, the organ cultures form anchoring sites and proliferative cell columns as well as EVT, detaching and migrating from distal sites of the columns [26e28]. Depending on the matrix EVT migrate on the surface (collagen I) or invade underneath the anchorage site (Matrigel) [29]. In accordance with invasion in vivo, EVT on Matrigel quit proliferation, loose their polarised morphology and upregulate invasion-specific proteins such as a1 integrin in distal areas of the explant (Figure 2). Similar to trypsin-isolated CTBs, variability between explant cultures requires a critical number of placentae and experiments, i.e. migration/invasion assays, in parallel. However, careful dissection and seeding of villi finally results in generation of pure EVT, which can be re-isolated from the matrix without stromal contaminations [30]. Isolated EVT can also be recultivated on ECM but remain in a nonproliferating state (J. Pollheimer and M. Kno¨fler, unpublished). SIGNAL TRANSDUCTION PATHWAYS MODULATING INVASIVE TROPHOBLAST DIFFERENTIATION Focal adhesion kinase (FAK) FAK is a widely expressed non-receptor protein tyrosine kinase (PTK) which plays a key role in growth factor- and integrin-mediated cell migration. The enzyme has been identified in focal adhesion contact sites which link ECM

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proteins to the cytoskeleton and promote migration by directed polymerisation/stress fiber formation of actin filaments at the leading edge of motile cells. Fibroblasts lacking FAK exhibit motility defects whereas elevated FAK activity was shown to be associated with tumour invasion and progression of cancer cells towards a malignant phenotype [31,32]. FAK is activated by clustering of integrins in focal adhesions as well as by growth factor- and G-protein-dependent receptor activation. Accordingly, the kinase contains regulatory protein regions interacting with integrin-associated proteins, paxillin and talin, but also contact sites binding Src-family members and other adaptor proteins containing Src homology (SH) domains [4]. Activation of FAK is achieved by phosphorylation at different amino acid residues. In particular, phosphorylation at Tyr-397 promotes high-affinity binding of Src-family PTKs resulting in activation of multiple proteins kinase cascades [33]. The event is thought to be critical during cell migration since increases in FAK phosphorylation at Tyr-397 selectively occurred at the leading edge of motile cells [34]. Switching of integrin expression has been noticed during invasion of isolated CTBs [35] as well as in differentiating explant cultures [29,30] suggesting that integrin-dependent events may fulfil an important role during invasive trophoblast differentiation. Indeed, a growth/invasion-promoting role of FAK and its phosphorylated form Tyr-397 has been demonstrated. The phosphorylated form of the kinase was predominantly detected in interstitial CTBs and was more abundant during the first weeks of pregnancy colocalising with the EVT-markers MMP-2 and a5 integrin [36,37]. Downregulation of FAK by adenovirus-mediated transfection with FAK antisense constructs or supplementation of FAKantisense oligonucleotides reduced outgrowth/migration of villous explant cultures and diminished invasion of isolated CTBs through Matrigel-coated chambers [36,37]. Treatment of HTR-8/SVneo cells with IGFBP-1 increased FAK phosphorylation suggesting that IGFBP-1-induced migration of the cells is partly achieved through FAK signalling [38]. Stimulation of putative EVTs, which have been passaged three to four times over a two week period with IGF-I, induced stress fiber formation and phosphorylation/localisation of FAK, paxillin and vinculin in focal adhesions [39]. G-proteins, RhoGTPases and Rho-associated kinase ROCK The identification of G-proteins (GTPases) was one of the major steps in understanding signal transduction since G-protein-dependent activation of adenyl cyclase/synthesis of cAMP upon hormonal stimulation was demonstrated [40]. Rho proteins comprise a family (RhoA, Racl, Cdc 42) of particular GTPases regulating diverse biological processes such as cell cycle, cellecell/focal adhesions, polarisation and cell migration [41,42]. Similar to the well known protooncogene Ras, the Rho-like GTPases function as molecular switches by cycling between an active GTP-bound and an inactive GDP-bound state [43]. Receptor stimulation by

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Figure 2. Extravillous trophoblast differentiation in villous explant cultures generated from first trimester placental tissue. A placental explant culture cultivated on collagen for 24 and 72 h was photographed at a 100-fold magnification (A, B). In addition, serial sections of an explant seeded for 24 h (C, D) and 72 h (E, F) on Matrigel are shown. Sections were immunohistochemically stained with specific antibodies and DAPI to visualise nuclei and photographed under the fluorescence microscope (200-fold magnification). Mesenchymal villi plated on collagen I proliferate during the early attachment period (A) and distally migrate at the matrix surface throughout the culture period (B). Invasion on Matrigel is accompanied by expression of proliferation-specific markers such as Ki67 in proximal areas of the cell column (C), whereas non-growing EVT precursors expressing p57KIP2 are formed in distal areas (D). EVT differentiation is accompanied by integrin switching since a6 integrin expression detectable in columns and villous CTB is downregulated in EVT (E) whereas a1 integrin is induced in the distal invasion zone (F). AT, anchoring tip, CC, cell column, EVT, extravillous trophoblast, VC, villous core, CTB, villous cytotrophoblast, S, syncytium.

Pollheimer and Kno¨fler: Signalling Pathways Regulating Invasive Trophoblast Differentiation

growth factors or celleECM interaction results in activation of Rho and generation of downstream events, i.e. formation of contractile actin bundles and rearrangement of microtubules [44]. RhoGTPases are controlled by guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and Rho-GDP dissociation inhibitors. Activities of Rho proteins are also dependent on FAK since active FAKeSrc signalling complexes regulate binding and phosphorylation of GAPs and Rho-GEFs [4]. Downstream effectors of Rho include p21-activated kinase (PAK), which crosstalks to the MAPK pathway by modulating Raf and Rho-associated kinase (ROCK). The Rho/ROCK signalling pathway promotes actin reorganization by, for example, activating c-Jun N-terminal kinase (JNK), which phosphorylates/activates transcription factors such as c-Jun or ATF2 [45]. Different Ga proteins modulating Ca2C levels and adenylyl cyclase activity were detected in trophoblasts [46]. Ras-GAP, a major downregulator of oncogenic Ras activity, is abundantly expressed in normal trophoblasts but downregulated in trophoblastic tumours and invasive moles suggesting that Ras activity is associated with trophoblast proliferation and invasion [47]. Signalling through inhibitory G-proteins was suggested to play a role in IGF-II-induced trophoblast migration. The growth factor was shown to diminish adenylyl cyclase activity and, in accordance, elevation of cAMP levels reduced basal migration of HTR-8/SVneo cells [48]. Functionality of the RhoA-ROCK signalling cascade has also been suggested during trophoblast migration. In situ, ROCK was detected in CTBs and syncytia, while RhoA was predominantly expressed in CTBs [49]. Presumptive EVTs used in functional assays were obtained after plating of minced first trimester placental fragments and additional six passages of the adherent cells. Treatment of these cells with selective Rho and ROCK inhibitors reduced spreading and migration through fibronectin-coated filters [49]. Mitogen-activated protein kinases (MAPKs) MAPKs control a wide range of biological processes including cell growth, development, inflammation, apoptosis and differentiation. The MAPK family comprises a large group of protein kinases which are activated through distinct molecular signalling pathways. Activation of extracellular signal-regulated kinases (ERKs) predominantly occurs through mitogenic stimuli such as growth factors and hormones whereas activation of c-Jun N-terminal kinase (JNK) and p38 MAPK is mainly achieved through stress stimuli and inflammatory responses [50]. A highly complex network of protein kinases regulates the activity of MAPKs through sequential phosphorylations at critical Ser, Thr and Tyr residues. Proteins of the MAPK kinase kinase (MAPKKK) family (Raf, MEKK, MLK, ASK1) phosphorylate MAPK kinases (MAPKK), i.e. MEK1, MEK2 (targets of Raf), MEK2, MKK3, MEK5, MKK7 (targets of MEKK), MKK3, MKK4, MKK7 (targets of MLK) and MKK3, MKK4, MKK6 (targets of ASK1) [51]. The MAPKK then

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activate the four major families of MAPKs, i.e. ERKs (targets of MEK1, MEK2), ERK5 (target of MEK5), JNKs (targets of MKK4 and MKK7) and p38 MAPK (target of MKK3 and MKK6) [52]. Classically, RTKs, GPCRs and integrins activate ERKs through the Ras ORaf O MEK cascade but other protein kinases such as PI3K can also modulate MEK activity [53e55]. Signalling through MAPK pathways results in activation of numerous transcription factors including members of the basic helix-loop-helix (bHLH), Ets-domain and bZIP protein families as well as different nuclear hormone receptors and zinc finger proteins [51]. Given the fact that MAPKs play a role in almost every cellular activity, it is not surprising that the enzymes also control cellular invasion and migration. ERKs, p38 MAPK and JNK were shown to regulate migration in different cell types using multiple mechanisms including phosphorylation of the focal adhesion adaptor paxillin [56], activation of myosin light chain kinase [57], upregulation of MMP-9 [58] and processing of MMP-2 [59]. Some growth factors use distinct MAPK cascades to elicit different responses. For example, FGFs induce cell proliferation through activation of ERKs whereas FGFdependent migration involves activation of p38 MAPK [60]. In the placenta, expression of ERK1 and ERK2 was detected in villous CTBs, but their active phosphorylated forms were only present until the 12th week of gestation suggesting a predominant role during early pregnancy [61]. In accordance with other cellular systems MAPKs were suggested to play a critical role in trophoblast growth and migration. Both IGF-II and IGFBP-1 induced migration of HTR-8/SVneo cells and phosphorylation of ERK1 and 2 [38,48]. Treatment of the cells with a specific MEK inhibitor reduced basal trophoblast migration and abolished the effects of IGF-II and IGFBP-1 [38,48]. Similarly, endothelin and EGF promoted HTR-8/SVneo cell migration which was accompanied by activation/phosphorylation of ERK1 and ERK2 [62,63]. Application of an MEK inhibitor also reduced HGF-induced but not basal motility of SGHPL-4 cells whereas inhibition of p38 MAPK had no effects [64]. Independent of growth factor signalling, the N-terminal fragment of uPA which lacks catalytic activity, stimulated migration of HTR-8/SVneo cells while blocking of MEKs reduced the effect [65]. The role of MAPKs in controlling proliferation of cell columns has not been tested, however, several growth factors controlling HTR-8/SVneo cell proliferation, i.e. TGF-a, amphiregulin or placental growth factor [66e68] likely act through ERK signalling. In addition, in choriocarcinoma cells lines angiotensin-II and vEGF modulate cell proliferation through ERK-dependent signalling [69e71]. BeWo cell proliferation is increased by leptin through ERK activation but this does not involve p38 MAPK [72]. Phosphoinositide 3-kinase (PI3K) PI3K is a major signalling component downstream of growth factor-activated RTKs and GPCRs [73]. Phosphorylated

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RTKs interact with p85 subunits of PI3K and recruit the enzyme to the membrane whereas GTPases activate PI3K through binding of its p110 subunit. At the membrane PI3K phosphorylates phosphatidylinositol-4, 5-bis-phosphate (PIP2) at the 3# position of its inositol ring and thereby converts PIP2 to PIP3. The Ser/Thr protein kinases Akt/Protein kinase B and PDK1 are activated through PIP3. Akt phosphorylates a wide range of other target proteins that control proliferation, survival and cell size [74,75]. PDK1 phosphorylates different protein kinase C (PKC) subunits. PKC as well as Ca/ calmodulin-dependent kinase II (CaMKII) are also controlled through the PIP3-mediated increase of cytosolic Ca2C levels. PIP3 levels are tightly regulated by lipid phosphatases such as PTEN (phosphatase and tensin homolog deleted on chromosome 10) converting PIP3 to PEP2. Activation of the PI3K/ Akt pathway and loss of function mutations of PTEN, which acts as a tumour suppressor, has been noticed in different cancers [73,76]. One of the critical targets of Akt is mTOR which plays a crucial role in PI3K-mediated oncogenesis [77]. The kinase mTOR controls cell cycle progression and cell size/mass through phosphorylation of proteins controlling protein translation, i.e. ribosomal S6 kinases [78]. Activation of PI3K plays a role of migration/invasion, for example during IGF-I-induced migration of vascular smooth muscle cells [79]. PI3K also plays a crucial role in growth factor-mediated trophoblast migration. Activation of PI3K with specific peptides resulted in increased motility of SGHPL-5 cells, whereas inhibition of PI3K reduced basal and HGF-induced migration [64]. Another growth factor promoting migration of isolated first trimester CTBs is EGF [80]. Recently, the integrated action of PI3K and ERK in EGF-stimulated phosphorylation and migration of HTR-8/SVneo cells was convincingly demonstrated [63]. The authors showed that the growth factor induced both ERK and Akt phosphorylation whereas EGF-activated cell migration could be blocked by inhibiting either MAPK or PI3K. In addition rapamycin, which specifically blocks mTOR, was shown to decrease phosphorylation of the 70 kDa S6 kinase and migration of the cells. Signalling proteins of the Smad family Smad proteins are the downstream effectors of the transforming growth factor-b (TGF-b) superfamily (TGF-b, activins, nodal, bone morphogenetic proteins) which regulate numerous cell functions, including proliferation, differentiation and production of extracellular matrix [81]. Signalling of the TGF-b family in mammals occurs through 5 type II and 7 type I Ser/Thr-RTKs, which are also known as activinreceptor-like kinases (ALKs), and each TGF-b member binds to a characteristic combination of type I and II receptors [82]. Autophosphorylation of the assembled receptor results in phosphorylation/activation of the receptor-regulated Smad proteins (R-Smads) Smad1, Smad2, Smad3 and Smad5 [83]. The antagonistic, inhibitory Smads (I-Smads) Smad6 and Smad7, compete with R-Smads for binding at the activated TGF-b receptors thereby inhibiting their phosphorylation

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[83]. The latter, however, form complexes with Smad4 which translocate to the nucleus and associate with different transcription factors modulating their activities in a positive or negative manner [83,84]. TGF-b factors were amongst the first identified regulators of invasive trophoblast differentiation since they inhibited proliferation of first trimester CTBs [17]. Furthermore, TGF-b exerts anti-invasive effects on trophoblasts by increasing tissue inhibitors of metalloproteinases (TIMPs) blocking MMP activity and by upregulating plasminogen activator inhibitor 1 (PAI-1) which inhibits uPA activity [85,86]. Conversely, antisense oligonucleotide-mediated inhibition of TGF-b3 expression in explant cultures promotes trophoblast outgrowth [87]. Contrary to TGF-b, activin promotes outgrowth and differentiation of villous explant cultures which was accompanied by expression of the EVTmarkers HLA-G and MMP-2 [26]. The expression and function of Smad proteins has not been investigated in trophoblast primary cultures, however, HTR-8 cells and the choriocarcinoma cell lines JEG-3 and JAR cells express Smad2, Smad4 and Smad7, whereas the latter lack Smad3 [88]. Upon TGF-b treatment of HTR-8 cells Smad3 was phosphorylated and translocated to the nucleus [88]. Ectopic expression of the Smad3 gene in Smad3-deficient JAR cells restored TGF-b-dependent PAI-1 and TIMP-1 expression but failed to reduce invasion in vitro suggesting that other mechanisms contribute to refractoriness to anti-invasive action of the cytokine [89,90]. Functionality of Smad proteins was further confirmed in JEG-3 cells. Expression of Smad2 and Smad4 enhanced TGF-b-stimulated expression of a luciferase reporter whereas expression of Smad7 inhibited reporter activity [91]. Other signalling cascades Various signalling cascades may also control invasive trophoblast differentiation, but have not yet been fully characterised. Cytokines activate the receptor-associated Janus kinases (JAKs) which induce phosphorylation, dimerisation and nuclear translocation of the signal transducers and activators of transcription (STATs) [92]. STAT proteins are not only involved in transmitting an inflammatory response but also play a critical role during cell movement [53]. STAT3, which plays a role in oncogenesis [93], can be detected by EMSA in choriocarcinoma cells and primary trophoblasts of early pregnancy, but not in third trimester trophoblasts suggesting a putative role in trophoblast invasion [94]. The NFkB signalling pathway has been recently implicated in controlling migration since PAI-1 has been identified as one of its targets [95]. Upon an inflammatory stimulus the IkB (inhibitor of kB) kinases (IKKs) phosphorylate IkB which is bound to the two subunits of NFkB (p65 and p50/p52) in the cytosol [96]. Phosphorylation of IkB results in its proteasomal degradation and translocation of NFkB to the nucleus. In villous explant cultures TNF-a inhibits trophoblast migration through EVT-specific induction of PAI-1 [30]. Recent evidence from our laboratory suggests that NFkB

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could be one of the mediators of PAI-1 induction since TNF-a stimulated binding of the factor to the PAI-1 promoter-specific enhancer element in EVT and HTR-8/ SVneo cells (S. Bauer and M. Kno¨fler, unpublished). Wnt represents another signal transduction cascade which plays an important role during cell proliferation, differentiation and oncogenesis [97]. In the canonical pathway engagement of Wnt with its receptors (dimers of LRP5/6 with frizzled molecules) results in inhibition of glycogen synthase kinase 3b (GSK-3b) which in the absence of a Wnt signal phosphorylates b-catenin resulting in its proteasomal degradation [98]. Wnt-dependent accumulation of b-catenin promotes its nuclear translocation and binding to the T-cell factor (TCP) family of transcription factors. The TCFs-becatenin complexes act as activators transcribing numerous target genes which control proliferation and migration/invasion [99e101]. In contrast, some ligands such as Wnt5A act in an antagonistic manner by promoting GSK-3b-independent b-catenin degradation and by activating the non-canonical Wnt pathway through Ca2C/calmodulindependent protein kinase II or JNK [102e104]. Analyses performed in our laboratory suggested abundant expression of TCF-3 and TCF-4 in EVT and trophoblastic cell lines (T. Loregger and M. Kno¨fler, in preparation). Moreover, a recombinant Wnt ligand modulated proliferation and migration of SGHPL-5 cells suggesting that the canonical Wnt/TCF pathway might play a critical role during invasive trophoblast differentiation. SUMMARY A wide range of well-studied signalling cascades controlling proliferation, invasion and migration of other cellular systems also play a role during invasive trophoblast differentiation. Activation of FAK as well as other integrin-dependent processes such as interaction with G-protein-mediated pathways regulate trophoblast invasion. Numerous growth factors acting predominantly through the ERK and PI3K pathways control proliferation and migration/invasion of trophoblast cell lines and isolated CTBs (summarised in Figure 3) but their final targets, i.e. executing transcriptional activators or repressors remain largely elusive. Whereas most studies were performed on isolated cell populations, the integrated signalling events controlling cell column formation, GO arrest at the distal cell column, detachment and migration/invasion are largely uncharacterised. A plethora of questions have not been fully answered and remain open for further investigations. For example, what is the role of villous stromal cells in EVT differentiation, what are the autocrine loops of EVT

Figure 3. Schematic presentation of signalling pathways stimulating trophoblast migration. Growth factors acting through these pathways are indicated. Stippeled arrows indicate signalling steps in individual cascades which have not been directly evaluated in trophoblasts, but in analogy to other cellular systems are likely operational. In general, growth factors evaluated so far signal through more than one pathway.

controlling proliferation, cell cycle arrest and differentiation, which mechanisms control the increase in polyploidy during EVT differentiation, what are the predominant signalling pathways regulating the process and can we identify trophoblast-specific signal transduction molecules/cascades? Some of these questions might be answered by using villous explant cultures of first trimester placentae, which similar to the in vivo situation perform column formation, proliferation, growth arrest, differentiation and EVT-specific marker expression in a temporal- and distance-related manner, but genetic manipulation of the system has to be improved. Careful analyses and comparison of different trophoblast model systems will help gaining more valuable insights into this exciting area of trophoblast research.

ACKNOWLEDGEMENTS Scientific work performed in M. Kno¨fler’s laboratory on trophoblast function and differentiation is supported by grant Nos. 8859, 9310 and 10719 of the Jubila¨umsfonds of the Austrian Nationalbank.

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