8th Postgraduate Course for Training in Reproductive Medicine and Reproductive Biology
Trophoblast differentiation and invasion: a lesson to be gained for understanding implantation of the human embryo
P. Bischof, A. Campana
Department of Obstetrics and Gynecology
Geneva University Hospital
Introduction
Despite the fact that some examples of viviparity exist in invertebrates, fish, amphibians and reptiles, implantation is a relatively new acquisition in evolution. The establishment of an intimate trophic connection between mother and embryo is a characteristic of mammals. Implantation is a new strategy in reproduction which allows the development of a small number of embryos in the protective maternal organism. This became possible with the establishment of a functional uterus. Although viviparity is an evolutionary advantage, it has one important limitation: the absolute necessity of a synchronisation between embryonic and uterine development.
Implantation in the human is unique (1). Mice, rats or rabbits are not suited to study human implantation. Guinea pigs have an intrusive type of implantation but syncytiotrophoblast forms before hatching. Even rhesus monkeys are different (superficial implantation, limited decidualisation, dual insertion at opposite poles etc...). This uniqueness is perhaps best illustrated by the fact that extrauterine pregnancies are not uncommon in humans whereas they are almost unknown in other mammals. Although human blastocysts resulting from in vitro fertilisation programs have been examined by conventional and electron microscopy, neither the stage of adhesion to the uterine surface nor the penetration phase have been observed in the human. Consequently the nature of these crucial events have to be deduced from information gathered in other primates (2) or from the very few observations gained by studying human blastocysts in vitro (3).
There is thus something very peculiar about human trophoblast. On one hand this drastically limits the use of animal models to study implantation and on the other hand the use of human embryos is impossible for obvious ethical reasons. One is thus left with developing in vitro systems with human trophoblastic cells.
Trophoblast differentiation
Human trophoblastic cells are easily isolated from placenta obtained either in early pregnancy (legal abortion products between 6 and 12 weeks of pregnancy) or after term deliveries. The use of term placental cells to study their invasive properties is less appropriate because the population of undifferentiated stem cells is less abundant at term as compared to first trimester cells (4).
Cytotrophoblastic cells (CTB) are derived from the trophectodermal cells of the blastocyst and represent a heterogeneous population during early pregnancy. After initial attachment of the blastocyst to the uterine lining, mononuclear CTB which surround the embryonic disc, fuse to form syncytia (5). These multinucleated terminally differentiated giant cells invade the pseudodecidualised endometrium (6). Once the definitive placental villi are formed, some CTB of anchoring villi (which contact the uterine wall) acquire a transiently invasive phenotype and invade the decidualised endometrium while the CTB of floating villi (in the extravillous space) remain attached to the villous basement membrane. Thus, CTB follow one of two existing differentiation pathways: Villous CTB (vCTB) form a monolayer of polarised epithelial stem cells which eventually differentiate by fusion to form a syncytiotrophoblast (STB, 5) covering the entire surface of the villous, or they can break through the syncytium at selected sites to form multilayered columns of non-polarised CTB. These motile and highly invasive extravillous CTB (evCTB, 7) also referred to as intermediate trophoblast (8) are found as cytokeratin positive cells in the decidua, the intima of uterine blood vessels and the proximal third of the myometrium. The molecular mechanism which directs CTB into one or the other differentiation pathway is the subject of intensive research in many centres.
Villous and extravillous CTB (vCTB and evCTB) are morphologically and functionally distinct (8, 9, 10). They are both cytokeratin positive indicating their common epithelial nature but differ in that evCTB express a non-classical human leukocyte class I antigen (HLA-G, 11,12), whereas vCTB express more epidermal growth factor receptors than evCTB (13). Endovascular CTB (enCTB), are a subclass of evCTB which invade the endometrial spiral arteries. Besides being cytokeratin positive (in contrast to endothelial cells which are vimentin positive) these cells quite uniquely express NCAM (14). Importantly, vCTB, evCTB and enCTB are in different microenvironments and thus surrounded by different extracellular matrix proteins (9).
Extracellular matrix components are known to influence adhesion, spreading, migration and differentiation of cells through specific cell surface receptors called integrins (15,16 for review). Integrins are heterodimeric transmembrane glycoproteins composed of a and ß subunits. Depending on the type of a ,ß combination the integrins bind to different matrix glycoproteins i.e. a5ß1 to fibronectin, a6ß1 to laminin etc. In the particular case of trophoblast, several studies (9, 17, 18, 19), including our own (20,21), have shown that vCTB and evCTB express different integrins. Villous CTB predominantly express the a6ß4 integrin (a probable laminin receptor, 21) polarised along the basement membrane. In contrast evCTB modulate their integrins. In the proximal region (CTB columns) they express a6ß4 in a non-polarised way whereas in the most distal part (the placental bed) they express the a5ß1 integrin, a Fn receptor. Thus, while CTB migrate from the villous into the decidua they modulate their integrin repertoire from being a6ß4 positive and a5ß1 negative to becoming a6ß4 negative and a5ß1 positive. Endovascular CTB express yet another integrin a1ß1 (23) a collagen receptor. These changes in integrin expression are linked with the acquisition of an invasive phenotype.
Purified and in-vitro cultured CTB undergo the same differentiation pathway and exhibit the same markers as in vivo. According to the culture conditions CTB can alter their phenotype in many ways. They can fuse and form STB (24), become evCTB and form cell columns (25,26), express HLA G (27), lose a6ß4 (26) and up-regulate a5ß1 and a1ß1 (23) and even invade a basement membrane preparation (23, 28-31). Clearly, human trophoblastic cells reproduce in vitro the differentiation pathways that they undergo in vivo even if they have been isolated from term placenta (32).
Metalloproteinase secretion
Invasion is not due to passive growth pressure but to an active biochemical process. A cell is invasive by virtue of its ability to secrete protease and CTB are no exception (29-31,33). Serine protease, cathepsin and metalloproteinase have been implicated in the invasive process (see 34 for review). MMP form a family of homologous enzymes which all have a Zn++ atom in their active site (34,35 for review) They are secreted as inactive pro enzymes (zymogens) which become activated upon partial hydrolysis whereby they lose their propeptide. They are classified in 3 subfamilies according to their substrate specificity: Gelatinases are represented by 2 enzymes, Gelatinase A and B (72 kDa and 92 kDa gelatinases or MMP-2 and MMP-9 respectively). These protease digest collagen type IV (the major constituent of basement membranes) and denatured collagen (gelatine). Collagenases include 2 protease the interstitial collagenase (MMP-1 or collagenase) and the neutrophil collagenase (MMP-8). These enzymes digest collagen type I,II,III,VII and X. They are thus appropriately designed for digesting the collagen of the extracellular matrix of the interstitium. Stromelysins is a subfamily of 4 enzymes MMP-3, 7, 10 and 11 (also called stromelysin-1, matrilysin, stromelysin 2 and 3 respectively). These protease have a relatively broad substrate specificity and digest collagen type IV, V, VII as well as laminin, fibronectin, proteoglycan and gelatine. Activation of the proMMPs into active MMPs can be reproduced in vitro by the addition of different agents such as mercurial salts. Although the physiological activators of the different MMPs are unknown, it has been shown that plasmin (36), MMP-3 (37) and membrane bound MMPs (MT-MMPs, 38-40) are potent activators of several MMPs. This means that MMPs act in cascade similar to the enzymes involved in blood coagulation. Direct evidence links the expression of MMPs, and particularly MMP-9 (41) to the metastatic phenotype, and the tissue inhibitor of metalloproteinases (TIMP) to the inhibition of metastatisation (42).
In vitro CTB invade an acellular amniotic membrane (43) or a reconstituted basement membrane (Matrigel 44,45), they thus behave like metastatic cells. This invasive behaviour is due to the ability of CTB to secrete MMPs since TIMP inhibits their invasiveness (47). Several studies have localised MMP proteins (48-51) and mRNA (49,50) in human trophoblast. Furthermore cultured CTB secrete MMPs (29-31,52) but CTB from early pregnancy are more invasive and secrete more MMPs than CTB isolated from term placenta (53). All MMPs are not equally important for trophoblast invasion. Gelatinase A and B (MMP-2 and MMP-9) have been shown to mediate CTB invasion into matrigel (31,47,54) but one must wonder if this is also true in vivo particularly since the nature of the matrix in which the cells are embedded plays such a crucial role in the regulation of MMP secretion (see below). Whatever the exact mechanism in vivo, one must admit that CTB behave like metastasis and that they secrete MMPs from very early in their development since human blastocysts (55) or even triploid 8-cell human embryos (31) produce MMPs.
Regulation of metalloproteinase secretion
Although CTB behave like metastatic cells, in vivo they are only transiently invasive (first trimester) and their invasion is limited only to the endometrium and to the proximal third of the myometrium (56). This temporal and spatial regulation of trophoblast invasion is believed to be essentially mediated by uterine factors. Of the many factors that have been shown to regulate the synthesis, activation or secretion of MMPs, we shall limit our discussion to only 2 types of regulators because of their clear endometrial origin: the extracellular matrix proteins and the cytokines.
Components of the extracellular matrix (ECM)
CTB cultured in the presence of fibronectin or rat tail collagen (collagen type I) flatten out, adhere to the culture dish and form non invasive syncytia (5). As discussed above, CTB have integrins which allow the cells to recognise their immediate environment and adapt to it. Collagen type I, the major component of the interstitium ECM has a remarkable stimulatory effect on gelatinase secretion by CTB (57, 58). Laminin, which promotes the invasive behaviour of melanoma cells and stimulates the secretion of MMP-2 (59) exerts a similar effect on BeWo choriocarcinoma cells but not on their non-malignant counterpart, the normal CTB (58). This indicates a certain degree of cell specificity. One study that directly implicates integrins in the regulation of MMP (60) showed that antibodies to the fibronectin receptor (integrin a5ß1) induce the synthesis of interstitial collagenase and stromelysin-1 (MMP-1 and MMP-3 respectively) in rabbit skin fibroblasts. Since fibronectin itself does not exert this effect, but fibronectin fragments do, the authors postulate that fibronectin (as well as other matrix proteins) have other domains, beside the cell recognition domain, which affect the cell’s decision to produce MMPs (61,62). This is probably also true for CTB since Irving and Lala (63) have shown that the fibronectin receptor, the integrin a5ß1, is essential for the migratory function of evCTB. Kliman and Feinberg (44) have convincingly shown that the ECM affects cell behaviour. CTB plated on matrigel respond differently depending on the thickness of the matrigel on which they are resting: If the matrigel is between 1 and 4 µm thick CTB adhere, aggregate and form syncytia, if it is between 4 and 14 µm thick CTB remain round, aggregate and invade the matrigel, with thicker matrigel matrices, CTB remain as mononuclear cells and do not invade the matrix. The mechanism leading to these altered responses remains to be explored.
The effects of cytokines
The literature on the effects of cytokines and growth factors on the invasive behaviour of cells is rather large and we shall limit our discussion to those cytokines which are either produced by the endometrium or for which the trophoblastic cells have receptors.
Interleukin-1 (IL-1) consists of 2 distinct but related peptides (IL-1 alpha and beta). IL-1, a known product of monocytes and macrophages is also produced by the tissues of the foeto-maternal interface. In the mouse, IL-1 is secreted by preimplantation embryos and IL-1 receptors type 1 (IL-1R-1) are expressed maximally by endometrial epithelial cells during the secretory phase thus at the time of implantation. When IL-1 receptor antagonists are given to mice prior to implantation, the number of implanted embryo is significantly reduced (64), implying that in mice IL-1 is an important mediator of implantation. In the human IL-1 is similarly distributed both at the protein and mRNA level (65,66).
Endometrial epithelial cells and extra villous but not villous CTB have IL-1R-1 both vCTB and evCTB but not endometrial epithelial cells produce IL-1. In the human endometrium IL-1 is produced by the decidualised or pseudo-decidualised stromal cells since its mRNA appears in these cells on the 23rd day of the cycle. IL-1 has been shown to stimulate the activity of MMP-1, MMP-3 and TIMP in human fibroblasts (67) and MMP-9 in CTB (68). The mechanism by which these stimulations occur involve protein kinase C and/or immediate early response genes (69,70).
Tumour necrosis factor (TNF) is a pleiotropic 14 kDa polypeptide produced by several cell types and particularly by macrophages (71). It is also produced by endometrial cells (72) and decidual macrophages (73) and TNF receptors have been characterised on human trophoblastic cells (74,75). Interestingly, TNF induces MMP-9 secretion in bovine endothelial cells (76) and MMP-1 and MMP-3 in human chorionic cells (77). But in contrast to IL-1, TNF decreases TIMP (77). In our hands TNF stimulates CTB MMP-9 but not MMP-2 (unpublished observation).
Epidermal growth factor (EGF)/Transforming growth factor alpha (TGFa) share amino acid sequence homology and bind to the same receptor. TGFa can be localised in the endometrium during the proliferative and the secretory phase but its expression is particularly high in decidual cells (78,79). The reported presence of TGFa in all forms of human trophoblastic cells (80,81) is probably an artefact (82) due to the massive expression of EGF/ TGFa receptors on CTB (81,82). EGF enhances the invasive behaviour of sarcoma cells (83) and CTB (82). This promotion of the invasive phenotype could be the result of either a direct stimulation of MMP-1 and MMP-3 as for fibroblasts (84) or a synergistic activation of these enzymes by IL-1 and EGF (85). It is clear from recent studies with CTB (86) that EGF does not alter the balance between MMP-2 and TIMP.
Leukaemia inhibitory factor (LIF) expression appears in the mouse endometrium on the 4th day of pregnancy just before implantation of the blastocyst (87). This transient expression of LIF is essential for pregnancy since in transgenic female mice lacking the LIF gene implantation does not occur. Furthermore, when the blastocysts of these transgenic mice are transferred to wild type pseudopregnant recipients, the blastocysts implant and lead to a normal pregnancy (88). The human endometrium also produces LIF (89) and LIF mRNA is more abundant in a secretory endometrium as compared to a proliferative one (90). LIF receptors have been found on both villous and extravillous CTB (91). LIF increases TIMP in human fibroblasts (92) and massively inhibits the gelatinolytic activity of CTB bearing a laminin receptor but not of CTB expressing the fibronectin receptor (94).
Transforming growth factor beta (TGFß) is represented by 5 homodimeric polypeptides which share 70 to 80 % structural homology. TGFß 1, 2 and 3 are produced by many mammalian cells. TGFß protein and mRNA have been localised in endometrial stromal, epithelial and decidual cells, as well as in villous and extra villous CTB and in STB (66,92,94,95). CTB have 3 types of TGFß receptors with differing affinities for TGFß1 and TGFß2 (96). In CTB or in human corneal fibroblasts, TGFß stimulates the synthesis of matrix glycoproteins such as laminin, fibronectin and collagen (97, 98). In human fibroblasts, in keratinocytes and in cervical cells TGFß increases MMP-2 and MMP-9 activity while it decreases TIMP (99-101). This however, is not the case for CTB because the inhibitory effect that decidual cell conditioned medium exerts on the invasive behaviour of CTB seems to be due to TGFß, since antibodies to this cytokine inhibit its effect (46,102). TGFß exerts this anti-invasive effect by stimulating the secretion of TIMP by CTB and inhibiting their migratory behaviour (63). Thus, TGFß could well be an endometrial signal which controls trophoblast invasion during implantation and placentation.
To conclude one could say that cytokines influence the secretion and or the activity of MMPs and although there is a certain degree of cell specificity, pro-inflammatory cytokines exert a stimulating effect whereas anti inflammatory cytokines are generally inhibitors of MMPs. Cytokines exert their effects mainly by inducing the transcription of immediate early response genes whose products act as transcription factors activating other genes, including those of MMPs.
The promoter region of the human MMP-9 gene
Since in our in vitro model MMP-9 activity is a prerequisite for invasion of matrigel by human CTB (31,47) and since many cytokines can dramatically regulate the expression of MMP-9 we have developed an interest in the promoter region of the human MMP-9 gene. The MMP-9 gene contains 13 exons and 12 introns for a total size of 7.7 kb (103). The regulatory region, (5’ flanking) of this gene was described in 2 studies (103,104) showing multiple cis-regulatory elements. These cis-regulatory elements are specific DNA sequences that bind trans-activators or trans-repressors (transcription factors) which are proteins encoded by other genes. Starting from the transcription initiation site (nucleotide 1) and making our way up-stream towards the 5’ end of MMP-9 promoter, one finds a TATA box and a retinoblastoma control element (RCE). RCE binds a protein called p105RB1 (the product of oncogene RB1). Other cis-regulatory elements are a TRE (phorbol ester, TPA, responsive element, see below), a NIP ( binds nuclear inhibitory proteins), a TIE (a TGFß inhibitory element), another TRE coupled to ets (ets binds ETS-1 and ETS-2, the products c-ets oncogenes). TRE-ets act synergistically to transactivate genes (105) and form a so called TORU (TPA and oncogene responsive unit). Further down stream there is another ets, an SP1 site (which binds the nuclear transcription factor SP-1) and an NFkB site (which binds p50-p65 heterodimers encoded by the c-REL oncogene family).
All cis regulatory elements are not equally important in the trans-activation of MMP-9. Indeed, transfection of HT 1080 fibrosarcoma cells have shown that the first TRE (-79 to -73) but not the second (-533 to -527) is essential but not sufficient for TPA (phorbol ester) or TNF induction of MMP-9 in (104). SP-1 and NFkB co-operate with TRE for a complete activation of the MMP-9 gene by TPA or TNF. In contrast, v-src (viral oncogene) activates MMP-9 gene transcription through the co-operation of TRE and RCE (106), in this particular case, SP-1 and NFkB are non functional.
Taken together this information points to an essential role of oncogene products as mediators of cytokine effects, as potent regulators of MMP-9 expression and possibly invasion.
Oncogenes and the invasive phenotype
Oncogenes
Oncogenes are genes that cause cancer. It now seems probable that the interplay between products of oncogenes is central to the development of most, if not all, cancers (107). Activation of oncogenes is thus a prerequisite for malignant transformation and acquisition of an invasive phenotype. Whether this is also true for trophoblast invasion remains to be investigated.
Oncogenes were first identified as virus genes capable of inducing tumours. It was later discovered that these viral genes are homologous to normal cellular genes (proto oncogenes) and highly conserved in animals. Since viral oncogenes can induce cancer, « activated » proto oncogenes also induce neoplasia. Activation occurs through different mechanisms such as mutations (due to carcinogens or radiation), gene amplification or chromosome rearrangement. Proto oncogene products can be classified into: cytokines, tyrosine kinases, receptors, G proteins, cell cycle regulators, DNA repair enzymes and transcription factors. They are thus responsible for essential cellular functions.
Transcription factors are nuclear proteins (sometimes also cytoplasmic from where they translocate into the nucleus) which bind DNA at specific sites located in the regulatory region of genes. Upon binding these proteins activate or repress the gene’s transcription machinery. The most widely known transcription factor is the AP1 complex (activator protein-1) initially described as a DNA binding protein which bound specifically to the enhancer element of SV40 (simian virus 40) and to the human metallothionein IIA gene (108 for review). It was later observed that all genes inducible by the tumour promoter phorbol ester TPA have a consensus sequence (TGAG/CTCA) known as the TRE site (TPA responsive element) which binds AP-1(109). The AP-1 complex is a heterodimer of Jun and Fos (108,110,111) the products of the proto oncogenes c-jun and c-fos which belong to the family of immediate early response genes. Jun and Fos can bind to each other in absence of DNA through their leucine zipper domains. Jun has a C terminal basic domain which recognises TRE. Jun’s binding to the DNA is regulated by several phosphorylation and dephosphorylation steps (112-115). Briefly phosphorylation of Jun in the N terminal transactivation domain increases whereas phosphorylation of the C terminal DNA binding domain decreases binding of AP-1 to the DNA. Thus when TPA acts on a cell by activating protein kinase C which phosphorylates AP-1 in the N terminus of Jun and/or phosphorylates an inhibitor (IP) of AP-1-TRE binding which becomes inactive. This results in an increased binding of AP-1 to TRE and leads to gene transcription. It is postulated that this is the mechanism by which TPA induces several metalloproteinases. As described earlier for the MMP-9 gene, AP-1 activation is sometimes necessary but not sufficient to trans activate a gene, and cross-coupling with other transcription factors (often NFkB or ets 116,117) bound to other DNA sites is necessary. The combined interactions between distinct classes of sequence specific transcription factors play an important role in regulating eukaryotic gene expression.
Oncogenes and MMP expression.
It is well documented that oncogenes induce an invasive behaviour. Transfection of ras oncogene in mammary epithelial cells transformed these cells into invasive cells capable of degrading a basement membrane (118). Clearly oncogenes can stimulate expression of basement membrane degrading enzymes. There are numerous reports showing that AP-1 is involved in the MMP-1 response to IL-1 (119,120), TNF (121), TPA (122,123), TGFb (119) and in the MMP-3 regulation by PDGF (124) and TGFb (125). TPA induces MMP-9 expression in HT1080 fibrosarcoma cells (100) and plating of rabbit synovial fibroblasts onto fibronectin induces MMP-9, a response which can be blocked by c-fos antisense (126). Although the promoter regions of the MMP genes are different, MMP-1, MMP-3 and MMP-9 but not MMP-2 have a TRE site capable of binding Fos-Jun heterodimers.
The AP-1 complex therefore occupies a key position in mediating signals that will lead to the acquisition of an invasive phenotype and to increased MMP expression. It is unknown so far if this is also true in trophoblast since in contrast to tumours, the invasive behaviour of CTB is acquired only transiently.
Oncogenes in trophoblast
The similarity between trophoblastic cells and transformed cells prompted several investigators to study the distribution of oncogenes in the human placenta (for review see 127). The two most widely studied oncogenes are c-erb B and c-fms, the products of which are the EGF and the M-CSF receptors respectively.
EGF receptors (EGF-R, 14,82,83,128,129) are predominantly found on villous trophoblast cells (CTB and STB), invasive evCTB lose their capacity to express EGF-R. This receptor which has an elevated expression in STB is thus considered as a marker of syncytium. Interestingly, the decidual cells massively express EGF-R.
M-CSF receptors (M-CSF-R 91,130-134) are already expressed at the blastocyst stage, then later by villous STB and evCTB but not by evSTB. Expression of M-CSF-R seems to be correlated with trophoblast invasiveness since this receptor is overexpressed in hydatidiform moles and BeWo choriocarcinoma cells and disappears when evCTB fuse to form syncytia.
The product of c-flt proto oncogene is a fms like tyrosine kinase which is the receptor of the angiogenic factor VEGF (vascular endothelial growth factor). This VEGF receptor is expressed in villous and extravillous CTB (135).
The oncogene c-sis encodes the beta chain of PDGF (platelet derived growth factor). This cytokine is expressed by early trophoblast (vCTB and evCTB) and by blastocysts (136-138). From the 6th week of pregnancy to term, PDGF decreases massively (137). Since PDGF stimulates the expression of c-myc (an oncogene involved in cell proliferation) and since these two products are coexpressed in cytotrophoblastic cells, it is believed that PDGF at least partially controls trophoblast proliferation (138).
Other oncogene products studied in the human placenta include pp60SRC (the product of c-src) a membrane associated tyrosine kinase (129) and c-kit, the stem cell factor (or Kit ligand) receptor (139). Unexpectedly we found only two reports on c-fos in the placenta (one in the human, 140 and one in the mouse, 141). Fos seems to be implicated more in trophoblast differentiation rather than in proliferation, since its mRNA is more abundant at term than in early pregnancy (140). To our knowledge c-jun has not been studied in the human trophoblast.
Tumour-suppressor genes have also been investigated in trophoblast. The protein p53 is a nuclear phosphoprotein involved in tumour progression. The wild type p53, an onco-suppressor, inhibits proliferation and promotes differentiation whereas the mutated p53 is oncogenic (142) In fact p53 mutation is the most common genetic change related to cancer. Overexpression of p53 inhibits oncogene induced transformation of cells, while the mutated form of p53 has lost this property and becomes oncogenic. It is interesting to note that the ability of p53 to inhibit proliferation is linked with its ability to down-regulate c-fos (143). These various observations make p53 an excellent candidate as a potential regulator of trophoblast invasion particularly since villous and extra villous CTB and villous STB express the wild type p53 (144-146) whereas the choriocarcinoma cell lines Jar, BeWo an Jeg express mutated p53 (145).
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