Fusion of Cytothrophoblast with Syncytiotrophoblast in the Human Placenta: Factors Involved in Syncytialization Gauster M, Huppertz B J

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Fusion of Cytothrophoblast with Syncytiotrophoblast in the Human Placenta: Factors Involved in Syncytialization Gauster M, Huppertz B J. Reproduktionsmed. Endokrinol 2008; 5 (2), 76-82

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Wir freuen uns auf Sie! Fusion of Cytotrophoblast with Syncytiotrophoblast in the Human Placenta: Factors Involved in Syncytialization M. Gauster, B. Huppertz

Human placental villi are covered by a characteristic epithelial-like layer. It consists of mononucleated cytotrophoblasts and an overlying syncytiotrophoblast layer both in contact to the trophoblastic basement membrane. The syncytiotrophoblast mostly lacks DNA replication and seems to transcribe only barely mRNA. Therefore, the syncytiotrophoblast depends on cell compounds delivered by fusing cytotrophoblasts. Delivery of fresh cytoplasmic contents into the syncytiotrophoblast is achieved by continuous fusion with cytotrophoblasts throughout gesta- tion. Fusion between cytotrophoblasts and the syncytiotrophoblast is driven by multiple factors, including environmental growth factors and cytokines, which turn on a specific cascade of fusogenic proteins in cytotrophoblasts destined for fusion. The cascade includes protein kinases and transcription factors, as well as induced expression of fusion-promoting proteins associated with the cell membrane. Additionally, specific proteases are activated, which cleave and remodel structural proteins to prepare the cell for fusion. However, not only fusogenic proteins, but also plasma membrane architecture and physicochemical factors such as calcium and oxygen affect intertrophoblastic fusion. Coordinated action of all factors involved is crucial for proper cytotrophoblast – syncytiotrophoblast fusion. Deregulation of a single factor might cause an inadequate fusion rate and could lead to pregnancy complications such as preeclampsia or even spontaneous abortion. J Reproduktionsmed Endokrinol 2008; 5 (2): 76–82. Key words: cytotrophoblast, syncytiotrophoblast, syncytialization, intertrophoblastic fusion

Intercellular Fusion Human Trophoblast trophoblast derived nuclei and other in the Human organelles, proteins and RNA as well Arising from the trophoblast of the as cytoplasm and membranes are In human, the phenomenon of cell- blastocyst, two trophoblast popula- transferred into the syncytiotropho- cell fusion occurs in several different tions differentiate early during gesta- blast. cell types. During fertilization, after tion. Extravillous trophoblasts leave penetration of the zona pellucida the basal membrane of anchoring Experiments using 3H-thymidine in- and entry of the sperm into the egg’s villi and invade the tissues of the ma- corporation revealed that DNA syn- perivitelline space, fusion of the egg ternal decidua basalis to attach the thesis does not occur in syncytio- and sperm cell membranes takes placenta to the uterus and to re- trophoblast, implying that syncytial place [1]. Macrophages can differen- model maternal spiral arteries. nuclei are unable to replicate [3]. In tiate and fuse to form multinucleated addition, 3H-uridine incorporation chondroclasts and osteoclasts [2], The second population is the villous experiments showed that the syncy- which are important for cartilage and cytotrophoblast, which upon specific tiotrophoblast is mostly lacking RNA bone development and remodelling. stimulation differentiate and fuse synthesis [4]. Taking in consideration Macrophages are also able to fuse with the covering syncytiotropho- that the syncytiotrophoblast does not into giant cells important in immune blast. The multinucleated syncytio- replicate and shows only little tran- defense. During embryonic develop- trophoblast in concert with the un- scriptional activity, the mechanism ment of skeletal muscle, mononu- derlying mononucleated cytotropho- of fusion with cytotrophoblasts be- cleated myoblasts fuse to form multi- blasts are located on a basement comes highly important. The integ- nucleated myotubes. Even in the membrane and constitute the villous rity of the syncytiotrophoblast de- adult skeletal muscle satellite cells trophoblast, the epithelial-like layer pends on continuous fusion and de- fuse with skeletal muscle fibers. In of the placental villous tree. Some of livery of cytotrophoblast derived cy- the human placenta, villous tropho- the cytotrophoblasts are trophoblast toplasmic contents throughout preg- blasts fuse to generate the character- progenitor cells and divide in an nancy. istic multinucleated syncytial layer, asymmetric way. While one daughter the syncytiotrophoblast. Extravillous cell retains its progenitor character, At the same time acquisition of fresh trophoblasts differentiate and fuse to the other is destined for differentia- cellular material requires deposition generate the trophoblastic giant cells tion. The final differentiation stage of of aged cytosolic content to keep characteristic for the decidua basalis a cytotrophoblast is fusion with the the biological balance of the syncy- during pregnancy. syncytiotrophoblast. This way, cyto- tium. Apoptotic material is packed in

Received: February 6, 2008; accepted after revision: May 14, 2008. From the Institute of Cell Biology, Histology and Embryology, Center of Molecular Medicine, Medical University of Graz Correspondence: Martin Gauster, PhD, Institute of Cell Biology, Histology and Embryology, Center of Molecular Medicine, Medical University of Graz, A-8010 Graz, Harrachgasse 21/VII, Austria; e-mail: [email protected]

76 J. REPRODUKTIONSMED. ENDOKRINOL. 2/2008 For personal use only. Not to be reproduced without permission of Krause & Pachernegg GmbH. tor (GM-CSF) have also been demonstrated to trigger syncytialization and hCG synthesis [7]. Moreover, transforming growth factor (TGF)-α and leukemia-inhibitory factor (LIF) have been suggested to promote syncytialization and production of hCG [8]. Interestingly, hCG itself, pro- duced by the syncytiotrophoblast can act as inducer of cytotrophoblast differentiation [8, 9]. In contrast, transforming growth factor (TGF)-β inhibits syncytial formation as well as secretion of hCG and hPL [10]. Also tumor necrosis factor (TNF)-α impairs syncytium formation of pri- Figure 1. Schematic representation of trophoblast differentiation within the villous trophoblast. mary term trophoblasts in vitro and Derived from trophoblast progenitor cells daughter cells start to differentiate and finally fuse represses hCG synthesis by down- with the overlying syncytiotrophoblast. Nuclei that have become an integrative part of the syn- regulation of β-hCG subunit expres- cytiotrophoblast change their morphology and display chromatin condensation. Finally late apoptotic syncytial nuclei are packed into syncytial knots and are released into the maternal sion [11]. blood stream. The arrows follow the route of cell types and nuclear changes during trophoblast differentiation. When environmental factors bind to their receptors on target tropho- membrane sealed vesicles – the so staining these cells with membrane blasts, mitogen-activated protein called syncytial knots – and is re- associated proteins such as desmo- kinases (MAPKs) may be activated leased from the apical cytoplasmic plakin or E-cadherin (Fig. 2). which in turn regulate complex pro- membrane of the syncytiotropho- grams of cell differentiation. Two blast into the maternal circulation. classical MAPKs, the extracellular Hence, the turnover of villous tro- Regulation of Trophoblast signal-regulated kinase1/2 (ERK1/2) phoblast in a placental villus is simi- Differentiation and p38, are implicated to play sig- lar to the turnover in stratified epithe- nificant roles in initiation of tropho- lia and includes proliferation, differ- Since cytotrophoblasts can either ac- blast differentiation and fusion. Stud- entiation, fusion and controlled require the extravillous (invasive path- ies employing specific ERK1/2 and/ lease of apoptotic material (Fig. 1). way) or the villous (syncytial path- or p38 inhibitors to primary tropho- way) phenotype, it is apparent that blast cultures revealed impaired To study the fusion process between differentiation has to be strictly regu- differentiation and syncytialization trophoblasts in greater detail in vitro lated. Factors that are involved in [12]. the BeWo cell model has attracted a governing these pathways are cyto- great deal of attention. The BeWo kines and growth factors derived Another potential key player in regu- cell line is a choriocarcinoma cell from the maternal and fetal environ- lating intercellular trophoblast fusion line, which shows intercellular fu- ment. The first growth factor de- is protein kinase A (PKA). Trans- sion when stimulated with forskolin scribed to induce syncytialization fection of the catalytic subunit of [5]. Treatment of BeWo cells with has been epidermal growth factor PKA into BeWo cells demonstrated this reagent leads to formation of (EGF) leading to secretion of the hor- that PKA activity is sufficient to in- multinucleated cells (syncytia) and mones hCG and human placental crease BeWo cell fusion [13]. Activa- an increase in the expression of hu- lactogen (hPL) [6]. Colony stimulat- tion of the PKA pathway led to a man chorionic gonadotropin (hCG). ing factor (CSF) and granulocyte- diminution of the desmosomal pro- Syncytialization can be visualized by macrophage colony-stimulating fac- tein desmoplakin and an increase of nuclei within syncytia. Stimulation Figure 2. A) Immunofluorescent staining of of PKA with either forskolin or cAMP human first trimester placenta (week 8). (both induce fusion) upregulated Mononucleated cytotrophoblasts (arrow) transcription of the transcription fac- show staining for E-cadherin in their apical and lateral cell membranes (red). The over- tor glial cell missing a (GCMa) [13], lying multinucleated syncytiotrophoblast which belongs to the family of zinc- (arrowhead) lacks lateral cell walls and is containing transcription factors [14]. negative for E-cadherin. The syncytiotrophoblast expresses hCG (green). B) and C) Immunofluorescent staining of forskolin and Initial overexpression experiments vehicle treated BeWo cells. Treatment of indicated a functional role of human BeWo cells for 48h with forskolin (20 μM) GCM in nervous system develop- induced formation of multinucleated syncytia, visualized by degradation of E- ment and gliogenesis in Drosophila cadherin (stained in red). hCG expression and mammals [15, 16]. However, in (green) was predominantly observed in syn- the human nervous system GCMa cytia of forskolin treated BeWo cells, but was not detected in significant was also rarely seen in mononucleated BeWo cells (treated and untreated). amounts, but was rather present in mammalian placenta [17, 18]. In hu-

J. REPRODUKTIONSMED. ENDOKRINOL. 2/2008 77 man placenta, expression of GCMa The question how morphology and mation [35]. In human trophoblast is restricted to a subset of villous physiology of the human placenta derived BeWo cells, knockdown of cytotrophoblasts and is implicated to would have proceeded without inte- CD98 expression by antisense and be associated with trophoblast differ- gration of retroviruses into the hu- siRNA techniques suppressed cell fu- entiation [19]. The involvement of man genome is rather speculative sion [34, 36]. Interestingly, treatment GCMa in trophoblast fusion is sub- and remains unsolved. However, the of BeWo cells with the fusion induc- stantiated by knockdown experi- fact that syncytin 1 is abundantly ing reagent forskolin led to an in- ments, revealing that specific GCMa expressed in placental trophoblast, creased expression of CD98 [37]. In targeting led to diminished intercel- but is suppressed by CpG methyla- the human placenta, CD98 was de- lular fusion in BeWo cells. GCMa tion in non placental cells [26] em- tected in cytotrophoblast and the knockout mice show defective pla- phasizes the important role of this syncytiotrophoblast [38]. The pro- cental labyrinth formation and em- protein in placental development. posed ligand of CD98 is galectin 3, bryonic lethality, implying an inte- The combined action of the two syn- which is expressed in epithelial and gral role of this transcription factor in cytins may well have been important immune cells [39]. Co-immunopre- placental development [20]. So far, for the evolution of the human pla- cipitation in BeWo lysates clearly two placental target genes are de- centa with syncytin 1 being crucial demonstrates binding of galectin 3 to scribed for GCMa. One is human for trophoblast fusion and syncytin 2 CD98 in vitro [37]. Galectin 3 is sug- aromatase [21] and the second is hu- displaying immunosuppressive ac- gested to bind to glycosylated sites of man syncytin 1 [22], which is sup- tivities [27]. CD98 by its carbohydrate recogni- posed to trigger membrane fusion. tion domain. Experiments with lac- Though syncytin 1 and 2 are factors tose, which binds with high affinity involved in placental syncytializa- to lectins, revealed an inhibition of Membrane Proteins Involved tion and immunosuppression, rare galectin 3 binding to CD98 and de- in Trophoblast Fusion and rather controversial information creased fusion in BeWo cells [37]. is available on their localization in Like CD98, galectin 3 was detected Though syncytialization is an event the human placenta. In situ hybridi- in both cytotrophoblasts and syncy- of membrane fusion, it is obvious zation detected syncytin 1 mRNA in tiotrophoblast of the human placenta that membrane proteins are involved the syncytiotrophoblast [23]. In con- [40]. in triggering the interaction of the trast, the syncytin 1 protein was lo- two different plasma membranes. calized in either in the syncytiotro- Gap junctional communication be- One membrane protein playing a phoblast alone [23], in the syncytio- tween cytotrophoblasts and the syn- key role in trophoblast fusion seems trophoblast and some villous cyto- cytiotrophoblast was shown to be to be syncytin 1. Syncytin 1 is a trans- trophoblasts [28], or in villous cyto- important for syncytialization [41]. membrane protein encoded by an trophoblasts and extravillous tropho- Gap junctions are composed of clu- envelope gene of an endogenous blasts [29]. Varying antibody clones, sters of connexin hexamers, which retrovirus of the HERV-W family staining protocols and tissue han- act as transmembrane channels. In [23]. When functional, it consists of dling may have given rise to these the human placenta, connexin 43 a transmembrane unit and an extra- discrepancies. (Cx43) was detected between cyto- cellular domain exposed on the sur- trophoblasts and between cytotro- face of the cell. Initial experiments So far, two receptors have been de- phoblasts and the syncytiotropho- revealed that overexpression of scribed for syncytin 1, the amino blast [42]. Studies with heptanol, a syncytin 1 in COS cells resulted in acid transporters ASCT1 and ASCT2 non specific junctional uncoupler the formation of multinucleated syn- [30]. They are sodium dependent blocking all connexin channels, dis- cytia [23]. In primary cytotropho- transporters of neutral amino acids played inhibited intertrophoblastic blasts, expression of syncytin 1 was such as alanine, cysteine and serine communication and a decrease in upregulated when cells were stimu- [31]. Both mRNAs were detected in syncytiotrophoblast formation [43]. lated with a cAMP analogue, which placenta, but ASCT1 expression was Antisense oligonucleotides targeted is known to induce fusion [24]. In faint [32]. The ASCT2 protein, also against Cx43 mRNA resulted in poor contrast, silencing of syncytin 1 in designated as type-D retrovirus cytotrophoblast fusion and de- primary trophoblasts inhibited syn- receptor (RDR) or amino acid trans- creased hCG secretion [44]. Hence, cytialization, substantiating an in- porter B0 (ATB0), was localized ex- Cx43 is directly involved in inter- strumental role of syncytin 1 in tro- clusively in the cytotrophoblast [33]. trophoblastic communication, differ- phoblast fusion [24]. entiation and fusion. Yet another putative amino acid Interestingly, screening of retroviral transporter, the CD98 antigen was sequences in the human genome for demonstrated to be important in tro- Proteases potential other viral envelope encod- phoblast fusion [34]. CD98, also ing genes revealed a second fuso- known as FRP-1 and 4F2, is a hetero- Remodelling of membranes and cy- genic gene product termed syncytin dimeric integral membrane protein, toskeletal proteins are crucial steps 2. Syncytin 2 is encoded by an endo- consisting of a heavy chain and a in the process of intercellular fusion. genous retroviral envelope gene, covalently attached light chain. The Proteins of the sub-membranous cy- which belongs to the HERV-FRD role of CD98 in membrane fusion is toskeleton are degraded by proteases family [25]. So far no fusogenic substantiated by the fact that its ex- to prepare defined areas of the properties have been allocated to pression is required for virus induced plasma membrane for fusion. Some syncytin 2. cell fusion, as well as osteoclast for- of the proteases involved in tro-

78 J. REPRODUKTIONSMED. ENDOKRINOL. 2/2008 phoblast fusion are family members ADAM12 is located in cytotropho- tral cholinephospholipids (e.g. phos- of the caspase family. The majority blasts and apical areas of the syncy- phatidylcholine, PC) are predomi- of caspases is basically known to tiotrophoblast in first trimester pla- nantly located in the outer leaflet, drive apoptosis, but recent studies centa. At term, immunohistochemis- negatively charged aminophospho- revealed key functions of such cas- try revealed positive staining of cyto- lipids (phosphatidylethanolamine, pases in cell differentiation [45–47]. trophoblasts [57]. However, immu- PE and phosphatidylserine, PS) are The term caspase (cysteine aspartase) nohistochemistry alone is not suffi- located in the inner leaflet [58]. The arises from the proteolytic behaviour cient to demonstrate a direct fuso- membrane asymmetry is actively of this protease family. Caspases are genic potential of ADAM12. Func- maintained by an aminophospho- cysteine proteases (use cysteine as tional fusion assays are pending to lipid translocase, which handles mis- the nucleophilic group for substrate elucidate this issue. directed PE and PS from the outer cleavage) as well as aspases (cleave leaflet back to the inner leaflet [58, the peptide bond C-terminal to as- 59]. These translocases (flippases) partic acid residues). In the human Plasma Membrane Architecture are inactivated by caspases [60] and placenta, caspase 8 is implicated to consequently the asymmetry can no play a key role in differentiation of Plasma membranes of human cells longer be maintained. Pronounced cytotrophoblasts and subsequent fu- are bilayers of asymmetrically dis- PS externalization to the outer leaflet sion with the syncytiotrophoblast [4, tributed phospholipids. While neu- was observed prior to fusion events 48]. When specific peptide inhibitors or antisense oligonucleotides Table 1. Factors that promote (+) or impair (–) fusion of trophoblasts. Evidence for involvement interfering with caspase 8 activity of listed fusion factors were provided either in BeWo cells (B), villous explants (VE) or isolated or transcription, respectively, were primary trophoblast cells (T). (?) indicates that functional fusion assays, demonstrating a role added to first trimester villous ex- of this factor in fusion, are pending. plant cultures, syncytialization was Growth factors, hormones and cytokines blocked [48]. Active caspases 8 and 10, which are activated by cleavage EGF growth factor + T [6] of their proenzyme states, were de- CSF growth factor + T [7] tected in a subset of differentiated GM-CSF growth factor + T [7] cytotrophoblasts [4]. Recently, cas- TGF-α growth factor + T [8] pase 14 was suggested to participate LIF cytokine + T [8] in trophoblast fusion. Forskolin hCG peptide hormone + T [8, 9] stimulated BeWo cells showed in- TGF-β growth factor – T [10] creased expression of caspase 14, TNF-α cytokine – T [11] while treatment with staurosporine and induction of apoptosis had no Protein kinases and transcription factors effect on caspase 14 expression [49]. ERK1/2 mitogen activated protein kinase (MAPK) + T [12] p38 mitogen activated protein kinase (MAPK) + T [12] Other candidate proteins facilitating PKA protein kinase + B [13] intercellular fusion are members of GCMa transcription factor + B [22] the ADAM (a disintegrin and a Mash-2 transcription factor – T [74] metalloproteinase domain) family. ADAM proteins contain disintegrin Membrane proteins and metalloproteinase domains and Syncytin 1 endogenous retroviral envelope protein + T, B [23, 24] many of them also comprise putative Syncytin 2 endogenous retroviral envelope protein ? [25] hydrophobic fusion peptides, which ASCT1 amino acid transporter ? [30–32] mediate cell-cell fusion [50, 51]. ASCT2 amino acid transporter + B [30–32] Two members of this family, fertilin α CD98 amino acid transporter + B [34, 36] and β (ADAM 1 and 2), were shown Galectin 3 lectin + B [37] to be involved in murine sperm-egg Connexin 43 gap junction protein + T [44] fusion [52]. In human, fertilins are not functional in fertilization [53]. Proteases However, other members of the Caspase 8 protease + VE [4, 48] ADAM family, the meltrins, were Caspase 10 protease ? [4] suggested to trigger fusion, as shown for meltrin α (ADAM12) in myoblast Caspase 14 protease ? [49] fusion [54] and osteoclast formation ADAM12 protease ? [57] [55]. In the human placenta two Membrane architecture splice variants of ADAM12 were de- PS flip externalization of PS to outer leaflet of tected by northern blots [56]. One membrane bilayer + B [63–65] long transcription variant, which in- ABCG2 xenobiotic/lipid transporter ? [67] cludes a transmembrane domain, was designated as ADAM12-L. The Physicochemical factors alternative short splice variant, hypoxia low oxygen tension – T, B [71–73] ADAM12-S, lacks the membrane an- calcium + B [78] chor and is the secreted isoform.

J. REPRODUKTIONSMED. ENDOKRINOL. 2/2008 79 such as myotube formation [61], tivity of transcription factors. Studies formation in a concentration de- membrane fusion of sperm and egg with isolated primary trophoblasts pendent way [76]. Considering the [62] and trophoblast syncytialization revealed an oxygen dependent effect fact that trophoblast fusion is medi- [63]. The assumption that PS exter- on the basic helix-loop-helix tran- ated by syncytin 1, which is an endo- nalization, the so-called PS-flip, is scription factor Mash-2 (mammalian genously expressed retroviral enve- a prerequisite of intertrophoblastic achaete/scute homologue 2) [74]. lope protein, it is tempting to specu- fusion was substantiated by in vitro While transcription of Mash-2 was late that trophoblastic syncytializa- experiments using monoclonal anti- down-regulated in primary tropho- tion requires extracellular calcium. phosphatidylserine antibodies. Fu- blasts cultured at 20 % oxygen, its BeWo cells, known for their fuso- sion efficiency of stimulated chori- expression was increased at 2 % genic potential, also require calcium ocarcinoma cells was decreased oxygen. The fact that overexpression for spontaneous, as well as forskolin when these antibodies were addi- of Mash-2 markedly inhibited tro- induced intercellular fusion [78]. Pri- tionally applied [64, 65]. However, phoblast fusion [74] substantiated mary cytotrophoblasts isolated from these antibodies do not recognize the inhibitory effect of low oxygen first trimester placenta remain in a phospholipids alone, but usually re- via Mash-2 on intercellular fusion. mononuclear state when cultured in quire a co-factor (e.g. beta-2-glyco- culture medium containing low cal- protein I) for sufficient binding [66]. Environmental calcium ions are re- cium concentrations [79]. Therefore, it is likely that protein- quired for fusion of artificial as well lipid complexes trigger syncytial fu- as biological membranes [75]. Vari- sion rather than PS externalization ous cell fusion experiments using Conclusions alone. cells overexpressing viral envelope glycoproteins emphasized the re- Fusion of cytotrophoblasts with the Recently, the xenobiotic/lipid trans- quirement of an appropriate extra- overlying syncytiotrophoblast is cru- porter ABCG2, a member of the ATP cellular calcium ion concentration cial for expansion, maintenance and binding cassette (ABC) family, was [76, 77]. Cells expressing the human functionality of the placental epithe- suggested to play a role in counter- immunodeficiency virus type 1 (HIV- lium-like layer, the villous trophob- balancing the increased PS externali- 1) envelope glycoprotein (gp120- last. An increasing number of factors, zation during trophoblast fusion gp41) and its receptor CD4 only involved in intertrophoblastic fusion, [67]. Indeed, ABC transporters have fused when calcium (millimolar con- emerged in the recent past (Tab. 1). been implicated to regulate phos- centration range) was supplemented They comprise proteins with diverse pholipid asymmetry by trafficking to the culture medium. In contrast, cellular functions, but also include structural lipids within plasma mem- the addition of calcium chelators “non-protein” factors such as cal- branes [68, 69]. ABCG2 was shown (EDTA, EGTA) inhibited syncytium cium concentration, oxygen tension to be up-regulated during trophoblast fusion [70]. Knockdown of ABCG2 with siRNA increased PS externalization and resulted in higher rates of apoptosis in forskolin induced BeWo differentiation [67]. Thus, ABCG2 was hypothesized to protect cells as survival factor during the period of asymmetry and reor- ganization of the plasma membrane during trophoblast syncytialization.

Physicochemical Factors

Several in vitro studies demonstrated an inhibitory effect of low oxygen tension on trophoblast fusion and differentiation. An oxygen concentration of approximately 9 % impaired fusion of isolated cytotrophoblasts and led to decreased hCG and hPL expression [71]. Forskolin stimulated BeWo cells, cultured un- Figure 3. Illustration of factors involved in trophoblast fusion. Environmentally derived growth der low oxygen tensions (2 % oxy- factors, hormones and cytokines bind to their cognate receptors at the plasma membrane of gen) showed suppressed intercellular cytotrophoblasts. Activation of either protein kinase A (PKA) or MAP kinases ERK1/2 and p38 leads to increased protein expression of the transcription factor GCMa, which in turn drives fusion [72], which was associated transcription of fusogenic genes. Several structural and membrane proteins were suggested to with relatively lower expression of promote trophoblast fusion, including syncytin 1 and its receptor ASCT2 (1), CD98 and its receptor syncytin and its receptor compared galectin 3 (2) as well as connexin 43 (3). Beside activation of PKA or MAP kinase pathways, to control conditions (20 % oxygen) cytokines induce conversion of pro-caspase 8 into active caspase 8. Once activated, caspase 8 can mediate inactivation of “flippases” and/or activation of “floppases” to trigger phosphatidyl- [73]. Additionally, hypoxia was dem- serine externalisation (PS flip) (4). Additionally, caspase 8 triggers remodelling of the sub-mem- onstrated to affect expression and ac- branous cytoskeleton by degrading structural proteins such as α-fodrin (5).

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