Human Reproduction Update, Vol.14, No.4 pp. 335344, 2008 doi:10.

1093/humupd/dmn010
Advance Access publication April 17, 2008

Trophoblast invasion: the role of intracellular cytokine

signalling via signal transducer and activator
of transcription 3 (STAT3)

Justine S. Fitzgerald, Tobias G. Poehlmann, Ekkehard Schleussner and Udo R. Markert1

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Klinik fur Frauenheilkunde und Geburtshilfe, Abteilung fur Geburtshilfe, Placenta-Labor, Friedrich-Schiller-Universitat Jena,
Bachstr. 18, 07743 Jena, Germany
1
Correspondence address. Tel: 49-3641-933763; Fax: 49-3641-933764; E-mail: markert@med.uni-jena.de, www.placenta-labor.de

Trophoblast cells display a very unique capability: they physiologically invade into the surrounding tissue. This capa-
bility is widely associated with tumours, and, indeed, the invasive behaviour of both is rather similar. The imposing
difference is that trophoblast cell invasion is temporally and locally controlled in contrast to unlimited tumour inva-
sion. It initiates immediately after embryo implantation into the endometrium. Parallel to tumours, trophoblasts
secrete proteases, such as matrix metalloproteinases, which dissolve the extracellular matrix and the surrounding
tissue. Thereby, these proteases prepare and allow true invasion of trophoblasts. The invasive capacities of tropho-
blasts are positively and negatively regulated by numerous cytokines including leukaemia inhibitory factor (LIF),
interleukin-6, hepatocyte growth factor, granulocyte macrophagecolony stimulating factor and others. They interact
via specific receptors with the trophoblast cells, in which they activate intracellular signalling cascades. These will then
induce expression of invasion relevant genes. One of these signalling pathways is the Janus kinase/signal transducers
and activators of transcription (STAT) pathway. Especially phosphorylated STAT3 enhances invasiveness of tumours
and trophoblast cells, where it is mainly activated by LIF. One of its most efficient physiological antagonists is
suppressor of cytokine signalling 3. The balance of these two intracellular molecules seems to be a key regulator of
tumour and trophoblast invasion.

Keywords: trophoblast cells; trophoblast invasion; STAT3; supressor of cytokine signalling 3; LIF

Introduction
Trophoblast cells are capable of invasion processes that are seen
also in malignancies with the exception that trophoblast cells are
physiological cells that grow invasively in a completely physio-
logical setting (Kliman, 1993; Koldovsky, 1999; Murray and
Lessey, 1999; Bischof et al., 2000) and, impairment of this type
of invasion can result in pregnancy related pathologies. Further-
more, trophoblast cells are able to mask or protect themselves
from the maternal immune system and thus escape potentially det-
rimental immunological responses stemming from the mother.
Cancerous tumour cells abuse and mimic these characteristics to
the disadvantage of the affected host. These two characteristics
make trophoblast cells exceptional in comparison to other physio-
logical cells in that they may pose as an exciting model in inves-
tigating the process of invasion. As the physiological and
pathological settings lie in close proximity to each other, the
deregulative progression may be better analysed. Especially intra-
cellular processes, such as signal transduction, are an attractive
field to explore since it seems that trophoblast cells themselves
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control their invasive growth in space and time (mainly limited
to the first trimester and the decidua) at least to some degree.
This may be seen in the situation of trophoblast cells originating
from extrauterine or xenogeneic gravidities since these cells are
just as capable of invasion as the trophoblast of regular pregnan-
cies (Randall, 1986; Poehlmann et al., 2004). Any boundary
seen here is usually found in physical limitations.
Another point of interest is the effects of local cytokines, pro-
duced either by trophoblast themselves or by other surrounding
cells (Fitzgerald et al., 2005a,b,c; Makrigiannakis et al., 2006).
These cytokines are often able to control trophoblast behaviour
in some way, by either modulating proliferation and migration
or inducing trophoblast to differentiate into an (non-)invasive phe-
notype. All of these cytokines use various intracellular signal med-
iating pathways. Some of these mechanisms have been explored
by several groups, but we are yet far from understanding the
whole picture. The signalling pathways of some cytokines are
still not completely illuminated. Exact mechanisms, particularly
in respect to possible crosstalk, are only partially understood.
335
Fitzgerald et al.

Early trophoblast development Implantation

The differentiation of the trophectoderm into two separate tropho-
blast subsets takes place immediately prior to invasion into the
decidual matrix, but seems to be triggered by the process of
adhesion (Armant, 2005). Initially, the outer trophoblast pheno-
type, the syncytiotrophoblast, invades the maternal endometrium
lining to create a cavity into which the blastocyst may embed
(Bischof and Campana, 1997). The capacity of syncytiotropho-
blast to invade the decidual matrix is lost after implantation. Fol-
lowing this period, cytotrophoblast, which are considered
trophoblast stem cells, supply a replenishment of trophoblast of
Implantation of the human blastocyst, and the event of trophoblast
invasion that accompanies this process, is considered by some to
be the major time limiting factor for the establishment of preg-
nancy (Herrler et al., 2003). Implantation begins with the apposi-
tion and attachment of the blastocyst to the uterine mucosa
(Fig. 1). At this time, the blastocyst is composed of a 5-cell
inner cell mass and surrounded by a 53-cell trophoblast hull, the
trophectoderm, which is destined to differentiate into the placenta.
Here, initial cell-to-cell contact is recognizable through the adher-
ence of the apical plasma membranes of trophoblast and uterine

the invasive phenotype, while the syncytiotrophoblast panel the
chorionic villi and intervillous space and take over a more endocri-
nological task. Trophoblasts existing outside of this intervillous
space are termed extravillous trophoblasts and have acquired the
invasive phenotype, in order either to anchor chorionic villi into
the Nitabuch layer or to profoundly infiltrate the decidua, reach
maternal spiral arteries and arrode them. The direction of invasion
seems to be determined through the expression of integrins of the
decidual matrix surrounding the trophoblast cell and the capacity
of this cell to produce matrix metalloproteinase (MMP) (Bischof
and Campana, 2000).
Trophoblast cells of the implanting blastocyst also participate in
a cyclic endocrinological process by producing human chorionic
gonadotropin (hCG), which in turn converts the corpus luteum
of the menstrual cycle to the progesterone producing corpus
luteum of pregnancy that promotes decidualization, protects grav-
idity, as well as supports the development of the embryo. Pro-
gesterone is involved in immunosuppression at the feto
maternal interface and thereby protects the fetus further from
unwanted immunological responses (Szekeres-Bartho et al.,
2005).
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epithelium (Enders and Mead, 1996). Normally, epithelial cells
do not allow adhesion of other cells to their apical surface
making trophoblast uterine epithelial attachment a unique cir-
cumstance (Hohn and Denker, 2002).
An excellent and quite detailed description of the implantation
window, as well as the development of the invasive trophoblast
can be found recently reviewed in this journal by Ferretti et al.
(2007).
The implantation window is understood to be the period of
maximal uterine receptivity, and is believed to commence 7
days after ovulation (around Day 20 of an idealized 28-day
cycle) and lasts no more than 2 days (Cavagna and Mantese,
2003). Several biological markers implicated in disclosing the
implantation window by dating the endometrium have attracted
attention in recent years (Fig. 1).
Invasion promoting cytokines
The focus of investigation has often been directed on the occur-
rence of cytokines during the complex event of placentation. It
is known that migration and invasion of extravillous trophoblast

Figure 1: Proposed scheme of implantation window and the role of LIF in fetomaternal crosstalk.
(1) Schematic representation of in utero free floating blastocyst surrounded by zona pellucida. At this time, blastocyst consists of an inner cell mass with a trophec-
toderm hull. Paracrine signalling (not depicted) probably attracts the blastocyst to putative implantation sites and synchronizes and orchestrates next steps, such as
(2) blastocyst hatching from zona pellucida. (3) Depicts gradual apposition of blastocyst to endometrium during the onset of the implantation window, delineated
here by two putative biomarkers for endometrial receptivity: pinopodes and LIF. LIF is maximally expressed by the endometrium at the time of implantation and the
blastocyst expresses the LIF receptor. In (4), the blastocyst adheres to the endometrium and then produces LIF itself, while, on the endometrium, the production of
gp130 and the LIF receptor increases. The concentration of soluble gp130 and the appearance of pinopodes on the endometrial surface are elevated at this time as
well. Adhesion induces trophoblast cells to differentiate into inner cytotrophoblast and outer syncytiotrophoblast layers as shown in (5), upon which the syncytio-
trophoblast invade into the luminal epithelium, where the blastocyst then commences to secrete cytokines such as IL-1, which in turn stimulates LIF expression in the
endometrium. In (6), implantation is complete, The implantation window is now closed. Hence, it is proposed that the embryo and the endometrium actively com-
municate through secretion of LIF and other cytokines in order to promote the complete implantation of the blastocyst.

336

cells are functionally controlled by a plethora of cytokines and
growth factors, but the relevance of individual cytokine-induced
signalling processes is still under elucidated.
One of the most important candidate pathways seems to be the
Janus kinase signal transducer and activator of transcription
(JAK STAT) cytokine signal transducing pathway. Several cyto-
kines that are important to reproductive medicine are known to
signal through this pathway in other cells. Below is a synopsis
of some of the most prominent of these cytokines and growth
factors, and their influence on the reproductive field (Table I).
Hepatocyte growth factor
The morphogenic hepatocyte growth factor (HGF) has been
described in mediating motility in tumours by activating STAT3
(a member of the JAK STAT signal transduction pathway, as
will be alluded to later). It seems that HGF is expressed by placen-
tal stromal cells, while the receptor is located on the trophoblast
(Saito et al., 1995). Furthermore, HGF is known to arbitrate the
invasive potential of trophoblast cells since trophoblast invasion
is dose-dependently increased by HGF (Kauma et al., 1999).
HGF knockout experiments in the mouse model have revealed
that expression deficiency leads to lethal placental failure due to
a complete lack of development of labyrinthine trophoblast
(Schmidt et al., 1995; Uehara et al., 1995). All of these facts
hint that HGF is of fundamental importance in the mesenchymal
induction of trophoblast growth and differentiation during the
development of the placenta (Stewart, 1996). Lack of HGF has
even been advocated in causing pre-eclampsia in human pregnan-
cies since the placentae from pre-eclamptic women were associ-
ated with a decreased HGF production (Kauma et al., 1999).
Finally, an altered expression of HGF and STAT3 has been
described in the placental tissues of malformed fetuses (Trovato
et al., 2002).
Interleukin-6
A further cytokine that utilizes STATs to mediate its signal is IL-6.
IL-6 is a cytokine that is correlated with a higher metastatic poten-
tial of cancer cells when found in the serum of cancer patients, at
least in the head and neck. IL-6 enhanced the invasiveness of head
and neck cancer cells (Nishino et al., 1998) and ovarian cancer
cells (Obata et al., 1997). In human choriocarcinoma, IL-6
failed to stimulate cell growth in cultures, but knocking out of
IL-6 resulted in inhibiting cell growth (Kong et al., 1996). More-
over, IL-6 found in the amniotic fluids of early pregnancy seems to
be produced by trophoblast cells (Jauniaux et al., 1996). This con-
centration could be positively correlated to gestational age. Cyto-
trophoblast cells, which are the invasive phenotype, express high
levels of IL-6 (Das et al., 2002) and increase the activity of
MMP-9 and MMP-2, both important proteins in invasion and
implantation (Meisser et al., 1999). Maximum IL-6 mRNA and
protein expression is identified in the human endometrium at the
time of implantation (Tabibzadeh et al., 1995; Vandermolen and
Gu, 1996) and its receptor is present on both maternal (endo-
metrial epithelial cells) and fetal tissues (trophoblast and blasto-
cyst) during implantation and placentation (Kojima et al., 1995;
Sharkey et al., 1995; Tabibzadeh et al., 1995). However, it
seems that IL-6-deficient female mice are fertile with normal
litter sizes and no significant difference in implantation rates
Trophoblast invasion and STAT3
similar to control groups (Bluethmann et al., 1994), although
IL-6 and LIF share gp130 as a common signal transducing mol-
ecule (see below). This cytokine induced an increased expression
of integrins associated with embryo attachment and its production
could be hemmed by hCG and progesterone (Das et al., 2002).
Reduced expression of IL-6 was found during the secretory
phase endometrium of women with recurrent miscarriage (Jasper
et al., 2007).
Interleukin-11
IL-11 is another cytokine of the IL-6 cytokine family and this
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conveys its signal through the same pathway. Female mice with
a null mutation of the IL-11 receptor a-chain are infertile
because of defective decidualization, which apparently leads to
uncontrollable trophoblast invasion (Bilinski et al., 1998; Robb
et al., 1998). The same receptor subunit has been detected on
developing decidual cells. IL-11 expression is at a maximum at
the time of decidualization in the human gravid uterus (Dimitriadis
et al., 2000). IL-11, its a receptor and LIF (see below) are dysre-
gulated in the endometrium of infertile women with endometriosis
during the implantation window (Dimitriadis et al., 2006a,b). Fur-
thermore, the production of IL-11 and decidualization are compro-
mised in endometrial stromal cells derived from patients with
infertility (Karpovich et al., 2005). These results are consistent
with the possibility of a paracrine mechanism between uterus
and decidua for STAT3 activation. As a matter of fact, a recent
study suggests that IL-11-induced signalling initiates and main-
tains the decidualization process through STAT3 and suppressor
of cytokine signalling (SOCS3) activation (Dimitriadis et al.,
2006a,b).
Concerning trophoblast cells, it could be demonstrated that the
IL-11 receptor is found on interstitial, or invasive/migratory, tro-
phoblast cells. Furthermore, this cytokine is able to boost the
migration of human trophoblast cells without altering the rate of
its proliferation and that this feat is probably accomplished
through stimulation of STAT3 phosphorylation (Paiva et al.,
2007).
Leukaemia inhibitory factor
LIF has been proposed to be indispensable during placentation in
several species (Stewart and Cullinan, 1997; Vogiagis and Sala-
monsen, 1999). LIF-deficient mice, though not sterile, are infertile
and fertility may be restored through infusion of LIF into the
uterus (Stewart et al., 1992). The fact that these LIF 2/2 blasto-
cysts are able to implant in pseudopregnant wild-type mice indi-
cates that maternal LIF deficiency prevents implantation in this
case. The blastocysts of LIF receptor knockout (KO) mice
implant, but die within 24 h of birth due to impaired placenta func-
tion (Ware et al., 1995). LIF is assumed to facilitate implantation
since LIF is present at high amounts in the maternal fetal inter-
face and because it is produced by both the human placenta and
endometrium (Cullinan et al., 1996; Kondera-Anasz et al.,
2004), where it is maximally expressed at the time of implantation
(Bhatt et al., 1991). LIF receptors are present on the trophoblast
(Kojima et al., 1995) and trophoblast cells are thus controllable.
During the implantation window, the embryo or blastocyst
expresses mRNA for the LIF receptor and then produces LIF
mRNA itself (Charnock-Jones et al., 1994; Sharkey et al., 1995;
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Fitzgerald et al.
Table I. Summary of mentioned cytokines, location and function in normal and pathological pregnancies.
Cytokine Location in human pregnancy
HGF Expressed by placental stromal cells
Receptor on trophoblast
IL-6 Probably produced by trophoblast in early pregnancy
Maximum expression in endometrium at implantation
Receptor on endometrial epithelial cells, trophoblast and
blastocyst (during implantation/decidualization)
IL-11 Maximum expression during decidualization
Receptor found on developing decidual cells
LIF Produced by placenta and endometrium
Maximally expressed during implantation window
Receptors on trophoblast and endometrial epithelial cells
GMCSF Synthesis in epithelial cells regulated by estrogen
van Eijk et al., 1996; Chen et al., 1999). When the blastocyst
invades the luminal epithelium and thus reaches the stroma, it
begins to synthesize cytokines such as IL-1, which in turn
induces LIF expression in the endometrium (Fig. 1) (Simon
et al., 1994a,b; Laird et al., 2000; Aghajanova, 2004).
Human choriocarcinoma cells increase regulation of HLA-G, a
non-classic class I MHC molecule believed to be involved in the
masking of the cell from the immune system, in response to
LIF (Bamberger et al., 2000). In addition, LIF could promote
the proliferation and invasion of choriocarcinoma cells and tro-
phoblast cells (Fitzgerald et al., 2005a,b,c). Knock down of
STAT3 in both cell types resulted in loss of LIF mediated invasion
(Poehlmann et al., 2005). It has been proposed that decidual
natural killer cells might regulate invading trophoblast cells as
they encounter them in the decidua by secreting LIF (Sharkey
et al., 1999). LIF suppresses its own effects by means of a negative
feedback mechanism on the JAK STAT pathway (Naka et al.,
1997). In this context, both too low and too high levels of LIF
in uterine flushings have been suggested to have negative predic-
tive value in implantation success (Laird et al., 1997; Ledee-
Bataille et al., 2002).
Granulocyte macrophagecolony stimulating factor
GM CSF, though not of the IL-6 type family, is also a mediator
that uses the JAK STAT pathway. GM CSF promotes DNA pro-
liferation, differentiation and secretory activity of human and
mouse cytotrophoblast cells in vitro (Athanassakis et al., 1987;
Armstrong and Chaouat, 1989) and thus probably functionally
supports the development of the placenta. Estrogen regulates the
synthesis of GM CSF by uterine epithelial cells in mice, sheep
and humans (Robertson and Seamark, 1992; Imakawa et al.,
1993; Giacomini et al., 1995). Even small amounts of exogenous
GM CSF administered to mice dramatically alters pregnancy
outcome (Chaouat et al., 1990; Tartakovsky et al., 1991). In
GM CSF-deficient mice, fetal growth and viability were jeopar-
dized due to compromised placental function. These effects
338
Effect of expression alternation in human reproduction
Placentas from pre-eclamptic women associated with low HGF production
Altered HGF expression in peri-implantation endometrium of excessive ovarian
responders after IVF treatment (implantation failure)
Reduced expression of IL-6 in secretory phase endometrium of women with
recurrent miscarriage
Dysregulated expression in endometrium during implantation window in
endometriosis patients
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Production and function compromised in endometrial stromal cells from infertility
patients
Dysregulated expression in endometrium during implantation window in
endometriosis patients
Too high and too low levels in uterine flushings have negative predictive value for
implantation success
Significantly reduced blood concentrations in pregnant patients suffering from
unexplained recurrent abortion
were increasingly deleterious when the conceptus was also
deficient, suggesting that GM CSF of either maternal or fetal
origin is required for optimal fetal growth and survival (Robertson
et al., 1999). One group could show that GM CSF concentration
in the peripherous blood of pregnant patients suffering from unex-
plained recurrent abortion was significantly reduced (Perricone
et al., 2003).
Regulation of invasion at the intracellular level
The JAKs STAT pathway
Regulation of invasion may occur not only on the extracellular
level, through cytokines, but also on the intracellular level. This
will be demonstrated on the example of JAK STAT signalling
pathway and the trophoblast cell and its malignant derivates, the
human choriocarcinoma cell.
Briefly (as illustrated in Fig. 2), receptors aggregate upon cyto-
kine binding, which leads to the juxtaposition of receptor associ-
ated tyrosine kinases called Janus kinases or JAKs (named after
the two-headed Roman mythological god, Janus, because of its
two-headed structure). These JAKs are now enabled to cross-
phosphorylate and activate each other, as well as specific
domains on the cytoplasmic domains of their respective cytokine
receptor. In doing so, intracellular STATs are now capable of
binding to these receptor domains, so the STATs may also in
turn be phosphorylated and activated by the JAKs. Upon acti-
vation, the STATs disassociate from the receptor and proceed to
form homo- and heterodimers that translocate into the nucleus
of a cell. Here, the STATs up-regulate or accelerate the transcrip-
tion of target proteins by binding and influencing the promoter
regions of these proteins. One of the transcribed proteins is the
SOCS protein, a regulator that is specifically and non-specifically
able to negatively modulate the duration of the cytokine signalling
response by binding to phosphotyrosine residues as seen for
instance on JAKs (Kamura et al., 1998; Hilton, 1999).
 Trophoblast invasion and STAT3

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Figure 2: JAK-STAT-Signal transduction pathway.
(1) Cytokine binding leads to receptor aggregation; (2) JAKs phosphorylate and activate each other and inner receptor ligand; (3) STATs bind to inner receptor
ligand to be activated by JAKs; (4) activated STATs disassociate to form homo-and heterodimers and translocate into nucleus to accelerate transcription of
target proteins; (5) one target protein family are SOCSs which limit further STAT activation by binding to JAKs.

Gp130, JAKs and STAT in knockout modelslessons
for reproduction
Disruption of gp130, the STAT3-activating subunit shared by all
members of the IL-6 receptor family, leads to an identical pheno-
type as knocking out of LIF (Ernst et al., 2001). It has also been
found that endometrial secretion of soluble gp130 (sgp130) is
increased multifold during the implantation window and that this
secretion appears to be reduced in infertile patients (Sherwin
et al., 2002). Interestingly, sgp130 blocks the biological activity
of LIF and IL-6 (Montero-Julian et al., 1997; Muller-Newen
et al., 1998; Zhang et al., 1998).
IL-6, and IL-6 type cytokines, leads to the activation of JAK1,
JAK2 and TYK2 (Heinrich et al., 1998). As reviewed by
Yamaoka et al. (2004), most Jak- or Tyk-KO mice are fertile
and produce viable phenotypes, but two interesting exceptions
are noted here. Jak12/2 mice are viable yet weigh 40% less
than their heterozygote littermates, and die perinatally due to
neurological deficits that encumbers suckling. A remarkable
insight is seen in the fact that the Jak1 KO mouse differs from
the phenotype observed in gp130 disrupted mice, but resembles
the phenotype of mice lacking the LIF-receptor subunit b (Rodig
et al., 1998).
Deficiency of Jak2 results in embryonic lethality at Day 12.5,
but this is due to a failure in erythropoiesis. No pathology concern-
ing the placentas of these pregnancies was reported (Neubauer
et al., 1998).
As Akira (1999) and Heinrich et al. (1998) beautifully review,
the effects of Stat deficiency in mice do not usually include

339
Fitzgerald et al.
impairment of fertility, implantation or embryo development, with
the exception of these two cases.
All of the above mentioned cytokines share STAT3 as a
common constituent of their intracellular signal transduction path-
ways and also seem to have a positive influence on the invasion,
proliferation and/or differentiation of trophoblast cells, thus facil-
itating pregnancy-related events, i.e. implantation.
Numerous reports indicate that STAT3 plays a role in reproduc-
tion. Evidently, as STAT3 is activated during the early post-
implantation period of the mouse and is essential for embryogen-
esis, STAT3 is indispensable to murine pregnancy. Wild-type
mouse embryos express STAT3 on the extra-embryonic visceral
endoderm 7.5 days post-coitum. Concurrent to this, STAT3
2/2 mice degenerate and die, but can be rescued through substi-
tution with an alternative splice form of STAT3, STAT3b, in
which the C-terminal transactivation domain is replaced with a
seven amino acid extension (Duncan et al., 1997; Takeda et al.,
1997; Akira, 1999). Furthermore, the inhibition of STAT3 acti-
vation in the endometrium of the mouse was shown to prevent
implantation (Catalano et al., 2005).
STAT5 consists of two highly related genes encoding STAT5a
and STAT5b, and was originally identified as a transcription factor
in mammary gland tissue whose phosphorylation is induced by
prolactin (Schmitt-Ney et al., 1992).
Especially STAT5b null mice showed signs of impaired fertility
as females consistently aborted between Days 8 17 of pregnancy
(Udy et al., 1997). In this study, no obvious maternal, placental or
fetal defect could be detected, but s.c. application of progesterone
maintained pregnancy to term. In a further study, the deletion of
STAT5a/b in mutant female mice resulted in infertility
(Teglund et al., 1998). Evidently, the cause of this infertility
could be distinguished in the ovaries of these animals, since,
although ovulation occurred normally, they were lacking in
large corpora lutea. The main site of STAT5a/b expression
appeared to take place directly in the corpora lutea and not in
developing or mature follicles.
Prolactin induces follicular granulosa cells to differentiate into a
functioning corpus luteum, in other words one that produces ade-
quate pregnancy-sustaining progesterone. In this context, it is
tempting to speculate that STAT5a/b-deficient infertility is due
implantation failure or placental insufficiency on the basis of pro-
gesterone shortage.
STAT3 in tumours
As mentioned earlier, the mechanistical similarity between inva-
siveness of trophoblast and cancer cells invites the sharing of
knowledge from one field to the other. The process of tumour inva-
sion involves the activity of signal mediators triggered by STAT3.
Aberrant activity of phosphorylated, dimerized STAT3 is advo-
cated to be causal for neoplastic cell behaviour, such as hyperpla-
sia, longevity or invasion, and thus for the malignancy of cells
(Bromberg et al., 1999). Indeed, constitutively activated STAT3
has been found in a number of tumours (Bowman et al., 2000).
STAT3 appears to play an essential role in the organization of
motility in various types of tumour and pluripotent cells (Boccac-
cio et al., 1998; Yeung et al., 1998; Zhang et al., 2002). Moreover,
STAT3 has been implicated in the transcriptional regulation of
proteases, a process with crucial importance for invasive cellular
340
growth (Smola-Hess et al., 2001; Puricelli et al., 2002; Udayaku-
mar et al., 2002).
Various reports indicate the influence of STAT3 on cellular
growth behaviour (reviewed in Buettner et al., 2002). Cell trans-
formation by aberrant STAT3 activity in tumours probably
involves up-regulation of genes promoting cell cycle progression
(cyclin D1, c-myc) and/or preventing apoptosis (bcl-xL; mcl-1;
survivin) (Bromberg et al., 1999; Epling-Burnette et al., 2001;
Shen et al., 2001).
STAT3 in trophoblast cells
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Using Jeg-3 human choriocarcinoma cells, as an easily controlla-
ble model for invasive, first trimester trophoblast cells, recent find-
ings suggest the possibility that STAT3 is a central player in
pathways triggered by diverse factors that modulate placentation
(Fitzgerald et al., 2005a,b). These findings indicate a causal con-
nection of LIF-driven STAT3 activity and an altered protease
expression pattern in Jeg-3 choriocarcinoma cells. Interestingly,
the two proteases whose mRNA levels were influenced by LIF-
dependent STAT3 activation have been described to contribute
to invasive cell behaviour or implantation: Tissue inhibitor of
metalloproteinase (TIMP)-1 expression was found down-
regulated in response to LIF, and caspase-4, formerly termed
IL-1b converting enzyme homologue 2 (ICH-2), was up-regulated
(Fitzgerald et al., 2005a,b). TIMP-1 is linked to inhibition of
metastasis (Bischof et al., 2001). In choriocarcinoma cells, its
expression was found to be reduced due to genetic changes and
this down-regulation was correlated with the hyperinvasive and
malignant phenotype (Lala et al., 2002). It is conceivable to
assume that TIMP-1 expression is directly influenced by
STAT3, since STAT3-driven TIMP-1 regulation was also
described in other cell types such as synovial lining cells and hep-
atocytes (Gatsios et al., 1996; Richards et al., 1997) and the
TIMP-1 promoter was shown to contain STAT3 recognition
elements (Richards et al., 1997). Caspase-4 generates the bioactive
form of IL-1b (Kamens et al., 1995). Its expression is low in all
human tissue except in ovaries and it is secreted by preimplanta-
tion mouse embryos. The importance of IL-1 processing by
caspase-4 in implantation is underscored by the fact that the
IL-1 receptor is maximally expressed in the endometrium during
the secretory phase and that the IL-1 receptor antagonist evokes
a lower rate of (murine) implantation (Simon et al., 1994a,b).
Immunoreactive IL-1b is present in the villous cytotrophoblast,
syncytiotrophoblast and intermediate trophoblast (Simon et al.,
1994a, b) and apparently suppresses, at least in endometrial
stromal cells, the expression of TIMP-1 mRNA (Huang et al.,
1998). Furthermore, TIMP-1 inhibits all MMPs in an activated
form, but preferentially binds to latent and active MMP-9
(Goldberg et al., 1992), which has been found to be critical for
cytotrophoblast invasion (Librach et al., 1991). Whether this
could be indicative of an autocrine regulation requires further
investigation.
Moreover, LIF promotes giant cell differentiation in vitro and in
vivo, with giant cells being murine epithelial trophectoderm cells
that have transformed into an invasive population at the time of
implantation. This differentiation is apparently contained
through negative regulation via SOCS3 proteins which suppress
the JAK STAT signal transducing pathway and is induced by a

broad spectrum of cytokines, including LIF (Starr et al., 1997;
Sutherland, 2003; Takahashi et al., 2003).
Regulation of STAT3
There are two splice forms of STAT3, a and b, whose potential
functional differences are currently the subject of intense research.
When both isoforms were overexpressed in Cos (monkey fibro-
blast) cells, only STAT3b was constitutively able to cooperate
with c-Jun, another oncogenic transcription factor (Schaefer
et al., 1995). On the one hand, STAT3b was described to be a
dominant negative regulator of STAT3a, on the other hand,
STAT3a-deficient mice can be rescued by STAT3b (Caldenhoven
et al., 1996; Yoo et al., 2002; Maritano et al., 2004). Interestingly,
a high activation level of STAT3b was observed in the HCC cell
line JAR in contrast to first trimester and term trophoblasts (Cor-
vinus et al., 2003). The role of STAT3b in choriocarcinoma will
have to be investigated, since it may provide the opportunity to
identify specific STAT3b target genes. It will be of particular
importance to characterize the mechanisms that limit and cease
STAT3 activity in trophoblasts. This regulation may occur via
above mentioned inhibitors of STAT signalling such as SOCS or
PIAS (protein inhibitor of activated STATs) (Hilton, 1999), by
receptor down-regulation or through the loss of cytokine secretion.
The dysregulation of STAT3 activity patterns during placental
development might provoke pathological processes such as
diseases related to defective placentation, hypoinvasion in
pre-eclampsia or neoplasms like human choriocarcinoma
(Tarrade et al., 2002). A better understanding of STAT function
during placentation may lead to novel molecular and pharmaco-
logical strategies to interfere with signalling processes (Buettner
et al., 2002).
SOCS and trophoblast
As reviewed by Boyle and Robb, the Socs3 KO mouse model
demonstrates that a lack of this gene leads to placental insuffi-
ciency, and ultimately, embryonic lethality around embryonic
Day 13 (Marine et al., 1999; Roberts et al., 2001; Takahashi
et al., 2003; Boyle and Robb, 2008). The highest rates for
SOCS3 expression could be found in cells surrounding the chorio-
nic plate, in labyrinthine, spongiotrophoblast and trophoblast giant
cells or in short, cells known amongst others for invasive capacity.
These embryos were rescued successfully upon supplementation
with wild-type extraembryonic tissues capable of SOCS3 compen-
sation (Takahashi et al., 2003, 2006). Interestingly, the defects of
Socs3-null placentae were counteracted by deleting the
LIF-receptor a-chain (Takahashi et al., 2003). Another group
was able to confirm that STAT3 mediates the constitutive
expression of SOCS3 (Isobe et al., 2006).
In the human, SOCS proteins, especially nuclear, were immuno-
localized to the syncytiotrophoblast (Keelan et al., 2003; Blumen-
stein et al., 2005). It has been postulated that decreased SOCS
activity in human gestational tissues may play a part in the onset
or progression of term, yet overexpression of SOCS in pre-term,
labour. Considering the fact that activated peripheral blood mono-
nuclear cells induce the phosphorylation of STAT1 and STAT3 in
human trophoblast cells (Jiang et al., 2007), it seems possible that
inflammatory cytokines as often found in infections causative to
Trophoblast invasion and STAT3
pre-term labour may be responsible for this elevation of SOCS
expression.
Conclusion
The current literature on functions of STAT3 in human trophoblast
cells indicates its involvement in regulation of invasiveness.
Several soluble factors which are generally present in the
decidua, mainly HGF, GM CSF, IL-6, IL-11 and LIF, have
been described to use JAK STAT pathways for signalling and
may thereby influence invasiveness. The knowledge about the
Downloaded from http://humupd.oxfordjournals.org/ at Uniwersytet Warszawski Biblioteka Uniwersytecka on May 23, 2015
role of the STAT3 negative regulator SOCS3 is very preliminary,
but an invasion controlling role may be expected.
Funding
The group is funded member of EMBIC (Embryo Implantation
Control; www.embic.org), an European Network of Excellence
within the 6th Framework Programme of the European Union
(Contract no. 512040).
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Submitted on August 23, 2007; resubmitted on February 4, 2008; accepted on
March 5, 2008