Trophoblast Signalling: Knowns and Unknowns –

A Workshop Report

M. Knoflera,*, S. R. Sooranna

b, G. Daoud

c, G. Stj. Whitley

d, U. R. Markert

e,

Y. Xiaf, H. Cantiello

gand S. Hauguel-de Mouzon

h

a Department of Obstetrics and Gynecology, Medical University of Vienna, AKH, Waehringer Guertel 18-20, A-1090, Vienna,Austria; b Imperial College of Science, London, UK; c Laboratoire de Physiologie Materno-Foetale, University du Quebec,Montreal, Canada; d Biochemistry and Immunology, St.George’s Hospital Medical School, London, UK; e Department ofObstetrics, Friedrich-Schiller-University, Jena, Germany; f Houston Medical School, University of Texas, Houston, Texas, USA;g Massaschusetts General Hospital and Harvard Medical School, Charlestown, MA, USA; h Case Western Reserve University,Cleveland, OH, USA

Paper accepted 4 February 2005

Keywords: Signal transduction; MAP kinase; Calcineurin; P13 kinase; PKA; Calcium; cAMP

Placenta (2005), Vol. 26, Supplement A, Trophoblast Research, Vol. 19doi:10.1016/j.placenta.2005.02.001

INTRODUCTION

Cells respond to different extracellular stimuli through a series

of signalling cascades. From receptor activation to biological

effect, each signal follows a pathway recruiting effectors and

adapters, a variety of proteins that interact with each other

generating a cascade of sequential steps. Some pathways are

linear, some are branched, some are linked to others to induce

specific or redundant events. However, most of the time

signalling molecules are common to several pathways, forming

a complex intracellular network.

The goal of the workshop was to delineate signalling

cascades governing trophoblast function and differentiation.

Trophoblast pathways that have been already characterised as

well as those that are just starting to be unravelled were

discussed. In particular, potential cross-talk between signalling

cascades and specificity of trophoblast signalling cascades were

included in the discussion.

MITOGEN-ACTIVATED PROTEIN

KINASE (MAPK) SIGNALLING

The general role of MAP kinases

MAPK signalling is universal in eukaryotic cells. The MAPK

cascade regulates diverse cellular activities such as mitosis,

metabolism, motility, migration, survival, apoptosis and

differentiation. As a general introduction, S.R. Soaranna

(Imperial College, London), reported on the mechanism of

* Corresponding author. Tel.:C43 1 40400 2842; fax: C43 1 404007842.E-mail address: martin.knoefler@meduniwien.ac.at (M. Knofler).

0143e4004/$esee front matter

MAPK activation and discussed different classes of MAPKs.

Engagement of receptor tyrosine kinases with growth factors,

interactions of integrins with extracellular matrix components

or recruitment of small GTP-binding protein such as the Ras/

Rho family in response to extracellular stimuli promote

activation of MAPKs [1]. To date four different families of

MAPKs were described, big-mitogen activated kinase 1 (also

known as ERK5), the extracellular-regulated kinases (ERKs),

c-Jun N-terminal kinases (JNKs), and p38 MAPKs, the latter

three being the most extensively studied groups in vertebrates.

Classically, ERKs are activated through mitogens, whereas

JNKs and p38 MAPKs are induced through stress response.

Each family of MAPKs is composed of a set of three

evolutionarily conserved, sequentially acting kinases: a MAPK,

a MAPK kinase (MAPKK), and a MAPKK kinase

(MAPKKK) [2]. Activation MAPKKKs, which are serine/

threonine kinases, leads to the phosphorylation and activation

of MAPKKs, which then stimulate MAPK activity through

dual phosphorylation on threonine and tyrosine residues

located in the activation loop of kinase subdomain VIII. Once

activated, MAPKs usually phosphorylate target substrates on

serine or threonine residues. The wide range of functions of

the MAPKs is mediated through phosphorylation of hundreds

of substrates, including phospholipases, transcription factors

and cytoskeletal proteins [3]. Activation of the MAPK

pathways occurs in all cell types but will be discussed with

respect to a representative tissue. In human myometrium,

MAPK signalling pathways are activated at parturition and has

been shown to occur after stimuli in vitro. Activation of

MAPKs alters downstream mediators and affects gene

expression by increasing the stability of mRNA (e.g. IL-8)

and the transcription of genes (e.g. Il-8 and PGHS-2) as well

� 2005 Published by IFPA and Elsevier Ltd.

S50 Placenta (2005), Vol. 26, Supplement A, Trophoblast Research, Vol. 19

as leading to the post-translation modification of many

proteins (cPLA-2). It is suggested that is a central process in

human parturition.

MAPK in trophoblast cell fusion

As an example of the role of MAPK in differentiation

processes, G. Daoud and Julie Lafond (University du Quebec

at Montreal, Montreal) reported on the putative role of

ERK1/2, p38 MAPKs and peroxisome-proliferator-activated

receptor g (PPARg) in trophoblast differentiation. ERKs and

PPARg have been previously implicated in syncytialisation

[4,5]. The data showed that secretion of human chorionic

gonadotrophin (hCG), as a marker of trophoblast differen-

tiation, was decreased upon incubation with either PD98059

or SB203580. The two compounds specifically inhibited

MEK (the MAPK phosphorylating ERK1/2) and p38

MAPK, respectively. The data also demonstrated that p38

pathway is highly solicited compared to ERK1/2 pathway in

differentiation process. Furthermore, ERK1/2 and p38 are

rapidly activated upon addition of FBS, but the activation of

p38 is delayed compared to ERK1/2. Interestingly,

inhibition of ERK1/2 pathway activates p38 pathway

and inversely suggesting a cross-talk between both pathways.

Therefore, the results suggest that both ERKs and

P38 MAPKs are involved in syncytium formation,

eventually using non-overlapping signalling pathways. In-

terestingly, PPARa ligands inhibit trophoblast differentiation

indicating a PPAR-isoform specific role on trophoblast

differentiation.

MAPK in trophoblast cell invasion

Similar to the fusion process, ERKs have been shown to play

an important role in growth factor-dependent regulation of

trophoblast invasion [6]. Guy Whitley (St. George’s Hospital

Medical School, London) reported novel results on hepatocyte

growth factor (HGF) and epidermal growth factor (EGF)-

dependent signalling which both promote trophoblast motility

and invasion [7,8]. The data showed that binding of HGF to

the c-Met receptor led to autophosphorylation of the receptor

and the recruitment downstream signalling molecules such as

phosphatidylinositol 3-kinase (PI3K) in trophoblastic

SGHPL-4 cells. Moreover, stimulation of the cells with

HGF induced rapid phosphorylation of ERK1 and 2 as well as

activation of the protein kinase Akt, which acts downstream of

PI3K [9]. Pharmacological inhibitors blocking MAPK and

PI3K activation resulted in the inhibition of both HGF-

stimulated trophoblast motility and invasion. Additionally,

evidence was provided that MAPK and PI3K pathways act

independently. Like HGF, EGF also activated PI3K, the rapid

phosphorylation of Akt as well as ERK 1 and 2 phosphory-

lation. Accordingly, blocking of PI3K or MEK inhibited

EGF-induced motility. However, unlike HGF, EGF stimu-

lated motility was not inhibited by rapamycin indicating that

activation of mTOR by Akt was not involved in the regulation

of cell motility by this growth factor.

SIGNALLING THROUGH SIGNAL

TRANSDUCERS AND ACTIVATORS

OF TRANSCRIPTION (STATS)

Another signalling pathway influencing trophoblast invasion is

the Janus kinase (JAK)eSTAT signal transduction cascade,

and in particular the STAT3 transcription factor. STAT3

activity correlates with tumour invasiveness and progression

[10]. In analogy, STAT3 binding activity was only detectable

in first trimester trophoblasts and choriocarcinoma cells but

absent from term placenta suggesting a role in trophoblast

invasion [11]. U.R. Markert (Friedrich-Schiller-University,

Jena) reported novel data on the role of STAT3 in trophoblast

invasion. Leukemia inhibitory factor (LIF) induced phosphor-

ylation and DNA-binding activity of STAT3 in JEG-3 cells.

Furthermore, an RNAi approach was utilised to knock down

STAT3 in choriocarcinoma cells and primary trophoblasts.

Application of STAT siRNA oligonucleotides reduced

spontaneous invasion of JEG-3 cells and first trimester cells

but also blocked LIF-dependent elevation of trophoblast

migration suggesting that STAT3 may play a significant role

in trophoblast invasion. Albeit the direct role of JAKs in

trophoblast migration has not been investigated so far,

activation of trophoblast STAT factors likely occurs through

the particular signalling pathway. A synergistic mechanism

between the MAPK pathway and SOCS was described [12],

but is still debated.

SIGNALLING THROUGH CALCINEURIN

AND NUCLEAR FACTOR OF ACTIVATED

T CELLS (NFAT)

Calcineurin is a protein phosphatase and a highly conserved

cellular signal transducer that couples different extracellular

signals to a variety of intracellular responses [13]. Y. Xia

reported on the role Ca2C-independent regulation of calci-

neurin signalling. The data showed that MEKK3/MEK5/

BMK1 MAP kinase pathway controlled calcineurin activity

through phosphorylation of modulatory calcineurin interacting

protein 1 (MCIP1) in human trophoblast cells. BMK1-

phosphorylated MCIP1 dissociates from calcineurin and binds

to 14-3-3, a cytoplasmic phosphoprotein binding protein. The

association of phosphorylated MCIP1 with 14-3-3 displaces

phosphorylated NFAT from its docking site on 14-3-3,

thereby allowing phosphorylated NFAT to serve as a substrate

for the activated calcineurin. Experiments using MEKK3-

deficient mouse embryo fibroblasts showed that MEKK3 is

essential in angiotensin II-induced calcineurineNFAT acti-

vation. The pathway may play a role in regulating trophoblast

differentiation, since angiotensin II-NFAT signalling was

recently demonstrated to inhibit trophoblast invasion [14].

SIGNALLING THROUGH CA2C

AND CAMP

Ca2C and b-adrenergic/adenyl cyclase signalling pathways

involving trimeric (Gs, Gi) G proteins have been identified as

Knofler et al.: Trophoblast Signalling S51

effector systems in the human syncytiotrophoblast [15].

H. Cantiello (Harvard Medical School, MA) reported on the

role of Ca2C and cAMP in the regulation of cationic channels

in the human syncytiotrophoblast. Recent studies by Cantiel-

lo’s group determined the presence of the Trp-type channel,

polycystin-2, and its contribution to Ca2C transport in this

epithelium [16]. Trp channels are ubiquitous transducers of

Ca2C-related cell activation in most non-excitable cells [17].

The new data suggested that despite their role as potential

targets of phosphorylation, some cAMP-dependent ion

channels, for which polycystin-2 may be a potential target,

may be controlled instead by kinase mediated phosphorylation

of associated cytoskeletal proteins. The findings indicated

a regulatory effect of the cAMP-dependent protein kinase A

(PKA) in Ca2C-permeable cation channels in syncytial

membranes. Experiments with the purified polycystin-2, the

channel responsible for this activity in the human syncytio-

trophoblast indicated that PKA may directly phosphorylate

the channel protein. Conversely, polycystin-2 mediated Ca2C

transport in turn could feed back in a regulatory mechanism

targeting local cytoskeletal structures associated with the

channels. The data suggested that PKA phosphorylation, in

concert with other regulatory signals may be an important

transducer of hormone-mediated control of cation transport

and electrical parameters in the human placenta.

CONCLUSION

Multiple signalling cascades control trophoblast differentiation

processes such as syncytialisation and invasion as well as

cellular functions including regulation of hormones synthesis

and transport mechanisms. Signalling through the kinases

MAPK, PI3K and PKA, discussed in the workshop play

a crucial role in several key activities of the trophoblast.

Despite the fact that an increasing number of signals operating

in placental cells are being unravelled, numerous open

questions remain to be solved. For example STAT3 or

NFAT, have been identified as regulators of trophoblast

invasiveness, however, the majority of downstream effectors of

trophoblastic signal transduction cascades have not been

elucidated. The hierarchy of integrated signals controlling

complex biological mechanisms such as adhesion, cell column

formation and migration are not understood. Furthermore,

developmental and differentiation-dependent changes in

signalling remain largely elusive. Experimental investigations

in human trophoblasts are hampered by the fact that

differentiation/function of particular signalling pathways

cannot be studied at all stages of placental development.

Further characterization of trophoblast models systems will be

helpful in studying the complex intracellular network of

trophoblast signalling cascades.

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