Placenta
Volume 30, Supplement , Pages 49-54, March 2009

Factors Involved in Regulating Trophoblast Fusion: Potential Role in the Development of Preeclampsia

Institute of Cell Biology, Histology and Embryology, Center of Molecular Medicine, Medical University of Graz, Harrachgasse 21/VII, 8010 Graz, Austria

Accepted 20 October 2008. published online 25 November 2008.

Article Outline

Abstract 

In the human placenta, turnover of villous trophoblast involves proliferation, differentiation and fusion of mononucleated cytotrophoblasts with the overlying syncytiotrophoblast. In this way the syncytiotrophoblast is continuously supplied with compounds derived from the fusing cytotrophoblasts. Acquisition of fresh cellular components is balanced by a concomitant release of apoptotic material as syncytial knots from the syncytiotrophoblast to the maternal circulation. In the turnover of villous trophoblast, fusion is an essential step and has been shown to be regulated by multiple factors, such as cytokines, hormones, protein kinases, transcription factors, proteases and membrane proteins. Dysregulation of one or more of these fusion factors entails aberrant fusion of the cytotrophoblast with the syncytiotrophoblast, which adversely affects the maintenance and integrity of the placental barrier. Unbalanced trophoblast fusion and release of apoptotic material into the intervillous space may provoke a massive systemic inflammatory response by the mother and thus lead to preeclampsia.

Keywords: Cytotrophoblast, Syncytiotrophoblast, Trophoblast fusion, Preeclampsia

 

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1. Preeclampsia 

Preeclampsia is a disorder restricted to pregnancy and the postpartum period that can cause maternal and perinatal morbidity and mortality [1]. In general, preeclampsia is defined by the occurrence of gestational hypertension associated with proteinuria after 20 weeks of gestation in a previously normotensive woman [1], [2]. The precise etiology of preeclampsia is still not clear, but it is generally accepted that the placenta rather than the fetus is responsible for its development. Despite various hypotheses on the etiology of this syndrome, the common factor that seems to stimulate the maternal response is villous trophoblast [3]. The discovery of early biomarkers to predict preeclampsia [4], [5] has confirmed that the villous trophoblast indeed plays a major role in the onset of maternal symptoms [6].

Several recent studies described the relation of preeclampsia to aberrant villous trophoblast turnover, which includes altered cytotrophoblast proliferation, differentiation and fusion of cytotrophoblasts with the syncytiotrophoblast. Here we recapitulate the available knowledge of factors involved in trophoblast fusion [7] and focus on the consequences of their potential dysregulation for the outcome of pregnancy.

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2. Human trophoblast 

Between days fifteen and twenty-one after fertilization, implantation of the blastocyst is followed by the villous stage of placental development. At this stage, trophoblasts start to differentiate along two differentiation pathways, to become extravillous (invasive pathway) or villous (syncytial pathway) trophoblasts. The subset of extravillous trophoblasts leaves the basal membrane of anchoring villi and invades the decidua basalis. There they remodel maternal spiral arteries and anchor the placenta to the uterus, finally reaching the myometrium of the placental bed.

The second population, the villous cytotrophoblast, remains at the basal membrane and is covered by the syncytiotrophoblast, a multinucleated layer without lateral membranes. After stimulation, villous cytotrophoblasts differentiate and fuse with the overlying syncytiotrophoblast. Previous experiments using 3H-thymidine incorporation revealed that DNA synthesis does not occur in the syncytiotrophoblast, indicating that syncytial nuclei are unable to replicate [8]. Additionally, 3H-uridine incorporation experiments showed that the syncytiotrophoblast has low rates of RNA synthesis [9]. A recent study employed antibodies against the actively elongating form of RNA polymerase II as well as antibodies against both active and repressed chromatin. The results suggested transcriptional activity in a proportion of syncytiotrophoblast nuclei. The same group performed fluoro-uridine incorporation assays with subsequent immunodetection of fluoro-uridine and found labeled nuclei in the syncytiotrophoblast, which substantiated their interpretation [10].

Since nuclei in the syncytiotrophoblast do not replicate and the transcriptional activity of this layer seems to be low, the mechanism of fusion of cytotrophoblasts with the syncytiotrophoblast becomes highly important. Syncytial fusion enables the transfer of cytotrophoblast derived nuclei and other organelles, proteins and RNA as well as cytoplasm and membranes into the syncytiotrophoblast. Permanent acquisition of fresh cellular components, however, requires continuous disposal of aged cytosolic content to maintain the homeostasis of the syncytiotrophoblast. Thus, apoptotic material is packed into syncytial knots at the apical plasma membrane of the syncytiotrophoblast, where these corpuscular structures are released as sealed membrane vesicles into the maternal circulation. Uncontrolled extrusion of apoptotic, aponecrotic and necrotic material, perhaps as a consequence of exaggerated cytotrophoblast–syncytiotrophoblast fusion, has been suggested to trigger preeclampsia [5]. Restricted fusion, in contrast, may result in depletion of fresh cellular components within the syncytiotrophoblast, leading to exhaustion of the syncytial layer. Hence, trophoblast turnover has to be regulated within a tight range, avoiding excessive as well as restricted cytotrophoblast–syncytiotrophoblast fusion.

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3. Physical process of membrane fusion 

Among the many hypotheses describing the physical mechanisms of membrane fusion, the stalk hypothesis is generally accepted [11], [12], [13]. According to this hypothesis, membrane fusion proceeds through a series of intermediate stages. In detail, approach of two membranes to close vicinity is followed by local perturbations of the lipid structure, merge of the proximal monolayers and formation of a stalk (hemifusion). The fusion process is assumed to proceed much faster than microseconds and is completed by expansion of the stalk and final formation of a pore [11], [12]. The stalk hypothesis is based on fusion of bilayers free of proteins. In contrast, fusion of biological membranes is much more complex, as membrane associated proteins participate in this process. Models for protein mediated membrane fusion exist [14], [15]. The proteinaceous fusion pore model indicates that oligomeric trans-membrane proteins form gap junction-like structures with central hydrophilic channels that open at the onset of fusion [14]. The scaffold model suggests that the proteins involved bring the membranes into close contact to enable the merger [15]. However, regulation of biomembrane fusion such as between cytotrophoblast and syncytiotrophoblast is not only governed by proteins directly involved in membrane fusion, but also by proteins preceding the intrinsic fusion process.

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4. Regulation of the syncytial pathway 

Cytokines and growth factors derived from the maternal and fetal environment are involved in regulating the syncytial pathway (Table 1). Epidermal growth factor (EGF) was reported to induce syncytialization of cytotrophoblasts and secretion of human chorionic gonadotropin (hCG) and human placental lactogen (hPL) in vitro [16]. Other factors described to promote syncytialization are colony-stimulating factor (CSF), granulocyte-macrophage colony-stimulating factor (GM–CSF), leukemia-inhibitory factor (LIF), transforming growth factor (TGF)-α as well as vascular endothelial growth factor (VEGF) [17], [18], [19]. Interestingly, hCG secreted by the syncytiotrophoblast can act in an autocrine manner to increase syncytium formation [19], [20].

Table 1. Factors that trigger (+) or impair (−) syncytialization of trophoblasts. Data were obtained by in vitro experiments using first trimester trophoblasts (FT), midtrimester trophoblasts (MT), term trophoblasts (TT), BeWo cells (B) or villous explants (VE). Some factors were suggested to play a role in trophoblast fusion, but evidence has not been provided so far (indicated by a question mark).
Growth factors, hormones and cytokines
EGFGrowth factor+TT[16]
CSFGrowth factor+TT[18]
GM-CSFGrowth factor+TT[18]
LIFCytokine+TT[19]
TGF-αGrowth factor+TT[19]
VEGFGrowth factor+FT, TT[17]
hCGPeptide hormone+TT[19], [20]
PL74 (MIC-1)Macrophage inhibitory cytokine 1+TT[25]
TGF-βGrowth factor−TT[22]
TNF-αCytokine−TT[21]

Protein kinases and transcription factors
ERK1/2Mitogen-activated protein kinase (MAPK)+TT[26]
p38Mitogen-activated protein kinase (MAPK)+TT[26]
PKAProtein kinase+B[27]
GCMaTranscription factor+B[31]
Mash-2Transcription factor−MT[61]

Membrane proteins
Syncytin 1Endogenous retroviral envelope protein+TT, B[32], [33]
ASCT1Amino acid transporter?−[35], [36]
ASCT2Amino acid transporter+B[35], [36]
CD98Amino acid transporter+B[37], [38]
Galectin 3Lectin+B[39]
Connexin 43Gap junction protein+TT[42]

Proteases
Caspase 8Protease+VE[9], [47]
Caspase 10Protease?−[9]
Caspase 14Protease?−[48]
CalpainProtease+TT, B[79]
ADAM12Protease?−[80]

Physicochemical factors
HypoxiaLow oxygen tension−TT, B[57], [59]
Calcium +TT, B[81]

Membrane architecture
PS flipExternalization of PS to outer leaflet of membrane bilayer+B[82], [83]

In contrast, tumor necrosis factor (TNF)-α as well as transforming growth factor (TGF)-β impaired syncytium formation of trophoblasts in vitro and inhibited secretion of hCG and hPL [21], [22]. TGF-β signaling is in part modulated by endoglin (Eng), which is an auxiliary receptor for several TGF-β superfamily members. The soluble form of Eng (sEng) is supposed to derive from proteolytic processing of the membrane bound Eng [23]. An elevated level of sEng in the maternal circulation has been discussed as a marker for the prediction of preeclampsia [24]. In addition, sEng was suggested to interfere with binding of TGF-β to its receptors and membrane bound Eng, which gave rise to dysregulation of TGF-β signaling. Since TGF-β counteracts trophoblast fusion, its sequestration by circulating sEng would lead to enhanced syncytialization and an unbalanced trophoblast turnover. However, this scenario can only be true if both s-Eng and TGF-β are present and active in the same compartment, the villous trophoblast. In contrast to TGF-β, PL74 (or macrophage inhibitory cytokine 1, MIC-1), a member of the TGF-β superfamily, was suggested to trigger trophoblast differentiation, since antisense PL74 transfection into term cytotrophoblasts led to inhibition of syncytialization [25].

When environmentally derived factors bind to their receptors on target trophoblasts, downstream pathways are switched on to initiate a complex program of cell differentiation. Two classical mitogen-activated protein kinases (MAPKs), the extracellular signal-regulated kinase1/2 (ERK1/2) and p38, were suggested to play significant roles in initiating trophoblast differentiation and fusion. Specific inhibitors for ERK1/2 and/or p38 impaired differentiation and syncytialization in primary trophoblast cultures [26]. Another potential key player suggested to regulate downstream processes of trophoblast differentiation is protein kinase A (PKA). Transient overexpression of the catalytic subunit of PKA was sufficient to increase cellular fusion of BeWo cells [27]. Administration of forskolin led to an up-regulated expression of the transcription factor glial cell missing a (GCMa) in BeWo cells [27]. GCMa belongs to the GCM family, a family of zinc-containing transcription factors [28]. GCMa was recognized as the first transcription factor involved in syncytialization of trophoblasts. Its expression in human placenta was shown to be restricted to a subset of villous cytotrophoblasts [29], which most likely were destined for fusion. Two target genes were described for GCMa in the human placenta. One is aromatase [30] and the second is syncytin 1 [31], which triggers membrane fusion.

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5. Factors that promote trophoblast fusion 

Syncytin 1, encoded by an envelope gene of an endogenous retrovirus of the HERV-W family [32], was shown to be up-regulated in primary cytotrophoblasts when cells were stimulated with a cAMP analogue known to induce fusion [33]. Knock down of syncytin 1 inhibited syncytialization of primary trophoblasts, substantiating an instrumental role of syncytin 1 in trophoblast fusion [33]. Interestingly, syncytin 1 expression is abundant in placental trophoblast, while it was shown to be suppressed by CpG methylation in non-placental cells [34]. The initiation of membrane interaction between fusing trophoblasts was suggested to take place by binding of syncytin 1 to the amino acid transporters ASCT1 or ASCT2 [35], [36].

The putative amino acid transporter CD98 (also known as 4F2 and FRP-1) was also suggested to be important for trophoblast fusion [37]. Knock down of CD98 expression by antisense and siRNA techniques suppressed cell fusion in BeWo cells [37], [38]. When binding of CD98 to its proposed ligand galectin 3 was blocked competitively, fusion in BeWo cells decreased [39].

Placental protein 13 (PP13) is another member of the galectin family [40], which has also been discussed as a marker for assessment of the risk for preeclampsia [41]. PP13 was shown to have weak phospholipase activity and sugar binding assays revealed strong affinity to saccharides such as lactosamines and glucosamines [40]. As shown for galectin 3 and CD98, trophoblast fusion could be facilitated via carbohydrate interaction between PP13 and a putative glycoprotein [40].

Beside the above mentioned membrane proteins connexins also were suggested to promote fusion of trophoblasts as shown for connexin 43 [42]. However, interaction of trans-membrane pore-forming proteins alone is certainly not sufficient for intercellular trophoblast fusion.

It is obvious that remodeling of the sub-membranous cytoskeleton as well as degradation of adhesion proteins have to occur before defined areas of plasma membranes can fuse. Specific proteases involved in degradation of cytoskeletal and cell adhesion proteins are caspases (cysteine aspartase) [43], which are well known for their role in apoptosis, but recently also attracted attention in terms of their function in terminal cell differentiation [44], [45], [46]. In the human placenta, caspase 8 was considered to play a fundamental role in differentiation of cytotrophoblasts and subsequent fusion with the syncytiotrophoblast [9], [47]. Syncytialization in first trimester villous explants was blocked in the presence of specific caspase 8 inhibitors or antisense oligonucleotides targeting caspase 8 protein expression [47]. Moreover, active caspases 8 and 10 were detected in a subset of differentiated cytotrophoblasts [9]. Recently, caspase 14 was suggested to participate in trophoblast fusion, since forskolin stimulation induced its upregulation in BeWo cells [48].

Other candidate proteins that were recently considered to facilitate trophoblast fusion were members of the ADAM (a disintegrin and a metalloproteinase domain) family. In addition to disintegrin and metalloproteinase domains, some members were shown to contain putative hydrophobic fusion peptides, which potentially mediate cell–cell fusion [49], [50]. Meltrins, also members of the ADAM family, were suggested to trigger fusion, as demonstrated for meltrin α (ADAM12) in myoblast fusion [51] and osteoclast formation [52]. In the human placenta one long transcription variant (ADAM12-L) and an alternative short splice variant ADAM12-S were detected [53]. However, ADAM12-S is secreted due to the lack of a trans-membrane anchor and its participation in cell fusion is rather unlikely.

A metalloprotease implicated to play a significant role in pregnancy is pregnancy-associated placental protein-A (PAPP-A), which increases in maternal serum during gestation and declines after delivery [54]. PAPP-A was detected in isolated trophoblasts and was demonstrated to increase during syncytiotrophoblast formation [55]. Whether PAPP-A promotes trophoblast fusion or its increased expression is just a consequence of syncytialization remains to be clarified. The fact that expression and secretion of PAPP-A were about 10 times higher in extravillous cytotrophoblasts than in villous cytotrophoblasts rather indicates a role in implantation and trophoblast differentiation processes other than fusion [56].

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6. Influence of oxygen on trophoblast fusion 

A growing body of evidence suggests an inhibitory effect of low oxygen tension on trophoblast fusion and differentiation [57]. Forskolin treated BeWo cells showed suppressed intercellular fusion when cultured under limited oxygen tension (2% oxygen) [58]. Furthermore, cultivation of BeWo cells under low oxygen concentration led to down-regulation of syncytin and its receptor compared to control conditions (20% oxygen) [59]. In this regard, transcription factors directly or indirectly controlled by oxygen were suggested to participate in regulation of trophoblast fusion.

Hypoxia-inducible factor-1 (HIF1) is one of the transcription factors that promote transcription in response to hypoxic conditions [60]. HIF-1 may form a heterodimer, composed of HIF-1α and HIF-1β and then binds to promoters of oxygen-responsive genes. A putative target gene of HIF-1 is the basic helix-loop-helix transcription factor Mash-2 (mammalian achaete/scute homologue 2), which was found to be up-regulated in primary trophoblasts under 2% oxygen and repressed when cultured at 20% oxygen [61]. Interestingly, overexpression of Mash-2 markedly inhibited trophoblast fusion [61], a fact that substantiated the inhibitory effect of low oxygen on intercellular trophoblast fusion via HIF-1 and Mash-2 regulation. Another popular target gene of HIF-1 is soluble vascular endothelial growth factor receptor-1 (soluble fms-like tyrosine kinase 1, sFlt-1), which binds VEGF and placental growth factor (PlGF) [62]. Increased serum levels of sFLT-1 potentially capture a bulk of free serum VEGF and could thereby impair the regulatory action of the growth factor.

The whole field of oxygen and its influence on trophoblast fusion needs to be approached with great caution since most of the in vitro studies have been performed using non-adequate culture conditions. The use of 20% oxygen as a standard control value of the oxygen concentration was challenged when Burton and Jauniaux clearly showed the in vivo oxygen concentrations within the placenta during and at the end of the first trimester of pregnancy were much less [63]. Thus there is the need for a detailed revisiting of the results rather than the conclusions of such studies.

At 10–12 weeks of gestation when the intervillous space opens to maternal blood the placenta is faced with a rapid increase of oxygen tension [64], which is accompanied by a burst of oxidative stress. During this critical phase, placental cells maintain their cellular homeostasis by activation of a specific bundle of enzymes that reduce reactive oxygen species. Pivotal to the antioxidant response is the transcription factor NF-E2 related factor 2 (Nrf2) [65], which binds to an antioxidant response element in promoter regions of genes expressed to protect from oxidative stress [66].

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7. Increased trophoblast fusion – a cause for the onset of the clinical symptoms of preeclampsia? 

Extravillous trophoblasts migrate from the trophoblastic cell columns and invade into the decidual-myometrial junction of the uterine wall. Here they induce the remodeling of the uterine spiral arteries. These physiological changes of vascular conversion are impaired in pregnancies complicated by intrauterine growth restriction (IUGR) and preeclampsia [67], [68]. Mechanisms like reduced proliferation, enhanced apoptosis or even increased fusion [69] of extravillous trophoblasts may well be responsible for the reduction of the number of invasive trophoblasts. It remains to be elucidated, whether the dysregulation of trophoblast invasion is caused by intrinsic (trophoblastic) or environmental (decidual, arterial) factors.

A well adjusted network of growth factors and cytokines seems to determine the fate of the villous cytotrophoblast. It is tempting to speculate that aberrant local concentrations of these regulators may provoke dysregulation of trophoblast turnover and finally may lead to the onset of preeclampsia. Though controversially discussed throughout the literature, there are certain growth factors and cytokines suggested to be linked to preeclampsia. GM–CSF levels were reported to be significantly higher in maternal blood and placental tissues of preeclampsia pregnancies [70]. PL74 was demonstrated to be overexpressed in placentas complicated by preeclampsia [25] and higher levels of LIF were detected in placental homogenates of preeclamptic compared with normotensive women [71].

However, not only do aberrant concentrations of growth factors and cytokines affect trophoblast turnover. The presence of receptor isoforms seems also to influence downstream signaling. The soluble receptor proteins sFlt-1 and sEng, which bind VEGF and TGF-β, respectively, were demonstrated to be elevated in the circulation of women who developed preeclampsia [72], [73]. These soluble receptors are suggested to catch circulating growth factors and impair their regulatory influence on trophoblast turnover. Impaired receptor activation obviously results in restricted downstream events. One of these downstream acting molecules is the transcription factor GCMa, which is downregulated in preeclamptic placentas [74] and gives rise to a decreased syncytin 1 expression [75], [76]. The fact that GCMa and syncytin 1 were demonstrated to be downregulated in placentas of women already suffering from preeclampsia may imply that under these conditions there is the need to downregulate trophoblast fusion.

Mayhew et al. [77] have shown that there are no differences between normal placentas and those from preeclamptic mothers in terms of exchange surface areas and diffusion distances. This data points to the fact that the overall volume of the villous trophoblast is not changed in preeclampsia. In 1991 Arnholdt et al. [78] showed that proliferation of villous trophoblast is increased during preeclampsia. It is tempting to speculate that under a constant trophoblast volume a higher proliferation rate leads to a higher fusion rate and a higher rate of trophoblast release. This goes in line with the fact that fusion promoting factors, such as the above mentioned growth factors are elevated even before the onset of preeclampsia, again suggesting that excessive trophoblast fusion may be a cause for the development of this syndrome.

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8. Conclusions 

Tightly controlled continuous fusion of villous cytotrophoblasts with the syncytiotrophoblast and release of apoptotic material from the syncytium is a requisite for the integrity of the placental barrier. Recent studies have suggested that various key players, such as cytokines, hormones, protein kinases, transcription factors, proteases and membrane proteins, are involved in trophoblast fusion (Fig. 1). Hence, fusion of cytotrophoblasts with the syncytiotrophoblast depends on multiple key players and dysregulation of some factors could result in aberrant villous trophoblast turnover. Dysregulation of a single factor may alter syncytialization of isolated cells or cell lines in vitro. However, such alterations of a single factor may be overcome in vivo by a variety of compensatory mechanisms.

  • View full-size image.
  • Fig. 1 

    Schematic representation of factors involved in trophoblast fusion (with permission from Gauster et al. [7]). Growth factors, cytokines and hormones from the fetal and/or maternal environment bind to their cognate receptors on the plasma membrane of villous cytotrophoblasts. This may lead to activation of cascades such as those involving protein kinase A (PKA) or MAP kinases ERK1/2 and p38 resulting in increased protein expression of e.g. the transcription factor GCMa, which in turn drives transcription of fusogenic factors. Such factors are mostly structural and membrane proteins that were suggested to promote trophoblast fusion. They include syncytin 1 and its receptor ASCT2 (1), CD98 and its receptor galectin 3 (2) and connexin 43 (3). Other pathways may be activated, too. One recently described pathway is the cytokine-induced conversion of pro-caspase 8 into active caspase 8. Once activated, caspase 8 may act to mediate inactivation of “flippases” and/or activation of “floppases” to trigger the externalization of phosphatidylserine from the inner to the outer leaflet of the plasma membrane (PS flip) (4). Furthermore, caspase 8 may trigger the reorganization of the sub-membranous cytoskeleton by degradation of structural proteins such as α-fodrin (5).

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9. Conflict of interest 

The authors do not have any potential or actual personal, political, or financial interest in the material or information described in this paper.

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Acknowledgements 

This work was supported by grants from the EU (Grant # 037244, project title Pregenesys) and the Franz-Lanyar-Foundation of the Medical University Graz, Austria (Projects # 321 and # 331).

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PII: S0143-4004(08)00353-6

doi:10.1016/j.placenta.2008.10.011

Placenta
Volume 30, Supplement , Pages 49-54, March 2009

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