1 Institut National de la Santé et de la Recherche Médicale, U427,
Paris, France
4 Laboratoire de Génétique Moléculaire, Faculté des
Sciences Pharmaceutiques et Biologiques, Université René
Descartes, Paris, France
2 Faculté des Sciences, Poitiers, France
3 Service d'Hormonologie, Hôpital Robert Debré, Paris, France
* Author for correspondence (e-mail: amalassi{at}pharmacie.univ-paris5.fr)
Accepted 2 May 2003
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Summary |
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Key words: Cx43, Placenta, Herv-W, hCG, Cell-cell fusion
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Introduction |
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Gap junctions are clusters of transmembrane channels composed of connexin
(Cx) hexamers. In general, the effects of Cx expression have been attributed
to gap junctional intercellular communication (GJIC) and sharing a common pool
of intracellular messengers and metabolites. Gap junctions provide a pathway
for the diffusion of ions and small molecules such as cAMP, cGMP, inositol
trisphosphate (IP3) and Ca2+. Connexins represent a
family of closely related membrane proteins, which are encoded by a multigene
family that contains at least 20 members in humans. These connexins have
different biophysical properties and functional and regulatory characteristics
(Willecke et al., 2002). The
permeability of junctional channels is finely regulated. This regulation
involves the cyclic phosphorylation and dephosphorylation of connexins and
changes in intracellular Ca2+, H+ and cAMP
concentrations. In addition, connexin expression varies during
differentiation, proliferation and transformation processes and following
treatment with biologically active substances such as growth factors and
hormones (Bruzzone et al.,
1996
; Kumar and Gilula,
1996
; Lau et al.,
1992
; Loewenstein,
1981
). The exchange of molecules through gap junctions is thought
to be involved in the control of cell proliferation, in the control of cell
and tissue differentiation, in metabolic cooperation and in spatial
compartmentalization during embryonic development
(Bani-Yaghoub et al., 1999
;
Constantin and Cronier, 2000
;
Lecanda et al., 1998
;
Loewenstein, 1981
;
Saez et al., 1993
).
We previously showed that Cx26, Cx32, Cx33, Cx40 and Cx45 are not detected
in human trophoblast, whereas Cx43 mRNA and protein are present between
cytotrophoblastic cells and between cytotrophoblastic cells and the
syncytiotrophoblast (Cronier et al.,
2002). Furthermore, in vitro studies using fluorescence recovery
after photobleaching (gap-FRAP) showed the presence of a functional gap
junctional intertrophoblastic communication before trophoblast fusion
(Cronier et al., 2001
;
Cronier et al., 1994
). In
addition, treatment of CT with heptanol (a nonspecific junctional uncoupler
blocking all connexin channels) inhibits trophoblastic GJIC leading to a
decrease in ST formation, which suggests a role for GJIC in trophoblastic
fusion (Cronier et al.,
1994
).
The possibility of nonspecific actions for heptanol lead us, using an antisense strategy, to determine the specific functional role for Cx43 in trophoblastic fusion and differentiation. We assessed the morphological and functional differentiation of cultured human villous trophoblasts by desmoplakin immunostaining, by measuring hCG production and by measuring the expression of trophoblast-specific genes (hCG and HERV-W). Furthermore, we used the gap-FRAP method to investigate functional GJIC.
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Materials and Methods |
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Modulation of gene expression by antisense oligonucleotides
Synthetic antisense oligonucleotides targeting Cx43 were purchased from
Biognostik (Gottingen, Germany). The phosphorothioate-modified Cx43 antisense
oligonucleotide is the reverse complement of a target sequence described
previously (Fishman et al.,
1991) (Table 1).
The absence of duplex and hairpin formations and the absence of
cross-reactivity with related sequences in GenBank were checked. The cells
were seeded into 35 mm dishes at a density of 100,000 cells per well, 4 hours
before the addition of phosphorothioate-modified antisense. Typically, normal
cultured cytotrophoblastic cells were incubated with 10 µM synthetic Cx43
antisense oligonucleotide and 10 µM scrambled antisense (control,
Biognostik). After 48 hours of incubation, the cells were harvested and
protein and total RNA were extracted.
|
Immunocytochemistry
To detect desmoplakin, cultured cells were rinsed with PBS, fixed and
permeabilized in methanol at 20°C for 25 minutes. A monoclonal
anti-desmoplakin antibody (1:400, Sigma-Aldrich, Saint-Quentin Fallavier,
France) was then applied, followed by fluorescein isothiocyanate
(FITC)-conjugated goat anti-mouse immunoglobulin (1:400, Jackson
Immunoresearch Laboratories, Wet Grove, PA), as previously described
(Alsat et al., 1996;
Frendo et al., 2000b
). After
washing, samples were mounted in medium with
4',6-diamidino-2-phenylindole (DAPI) for nuclear staining (Vector
laboratories, Burlingame, CA).
To detect Cx43, specimens were fixed for 10 minutes in methanol at
20°C. They were then washed three times in PBS and processed using
a method similar to that described by Tabb et al.
(Tabb et al., 1992). After
incubation in a blocking solution consisting of 2% bovine serum albumin and 1%
Triton X100 in PBS for 30 minutes at room temperature, specimens were washed
three times in PBS and incubated overnight with monoclonal Cx43 primary
antibodies (1:200, Transduction Laboratories, Lexington, KY). After five
further washes in PBS, FITC-conjugated goat anti-mouse antibodies (1:100) were
applied for 45 minutes at room temperature. After washing, samples were
mounted in medium with DAPI for nuclear staining. The controls, which
consisted of omitting the primary antibody or applying the nonspecific IgG of
the same isotype, were all negative.
Gap-FRAP method
The degree of intercellular communication between neighboring cultured
trophoblastic cells was determined by measuring the cell-to-cell diffusion of
a fluorescent dye (Wade et al.,
1986) using an interactive laser cytometer (ACAS 570, Meridian
Instruments, Okemos, MI). Briefly, the cells were internally loaded for 10
minutes at room temperature with the membrane-permeant molecule,
6-carboxyfluorescein diacetate (7 µg/ml in 0.25% DMSO; Sigma Chemical Co.).
The highly fluorescent and membrane impermeable 6-carboxyfluorescein moiety is
released and accumulates within cells. After washing off the excess
extracellular fluorogenic ester to prevent further loading, a cell adjacent to
other cells was selected and its fluorescence was photobleached by strong
laser pulses (488 nm). Digital images of the fluorescent emission excited by
weak laser pulses were recorded at regular intervals for 12 minutes (scanning
period 2 minutes before and after photobleaching) and stored for subsequent
analysis. In each experiment, one labeled, isolated cell was left unbleached
as a reference for the loss of fluorescence due to repeated scanning and dye
leakage, and an isolated, bleached cell served as a control. When the return
of fluorescence followed a fast step-like course, reaching
90% of the
final steady state within <30 seconds of photobleaching, the diffusion of
the dye was neither prevented by the cell membranes nor limited by the
presence of gap junctions. We thus assumed that the fusion of cell membranes
had been completed and that the cellular elements were part of a true
syncytium. When the bleached cells were connected to unbleached contiguous
cells by open gap junctions, the fluorescence recovery followed a slow
exponential time-course. Therefore, the analysis of the kinetics of
fluorescence recovery makes it possible to distinguish between aggregated
cytotrophoblastic cells and the syncytiotrophoblast. In our experimental
conditions, GJIC was investigated (coupled cells or not) by measuring the
percentage of coupled cells in a population of neighboring cells. GJIC was
analyzed 2 days after plating. Three different topologies were recognized:
contiguous cytotrophoblastic cells, contiguous syncytiotrophoblasts, and
contiguous cyto- and syncytiotrophoblasts. During trophoblast differentiation
and cell treatments, the percentage of coupled cells was analyzed whatever the
topology of the trophoblastic elements
(Cronier et al., 1994
).
Syncytium formation analysis
Syncytium formation was followed by fixing and immunostaining cells so that
the distribution of desmoplakin and nuclei in cells could be observed
(Keryer et al., 1998). The
staining of desmoplakin present at the intercellular boundaries in aggregated
cells progressively disappears as the syncytium is formed
(Alsat et al., 1996
;
Douglas and King, 1990
). The
nuclei contained in 100 syncytia in a random area near the middle of the
slides were counted. Three coverslips were examined for each experimental
condition. Results are expressed as number of nuclei per syncytium.
Hormone assay
The concentration of hCG was determined in culture media by use of the
chemiluminescent immunoassay analyser ACS-180SE system (Bayer Diagnostics,
Germany). The sensitivity of the assay was 2 mU/ml. All values are
means±s.e.m. of triplicate determinations.
RNA isolation and analysis
Total RNA was extracted from cultured cells as described by Qiagen
(Courtabeuf, France). The total RNA concentration was determined at 260 nm and
its integrity was checked in a 1% agarose gel. The relative mRNA levels of the
different genes were measured by quantitative reverse transcriptase-polymerase
chain reaction (RT-PCR), essentially as previously described
(Frendo et al., 2000a), using
an ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystem,
USA) and the SYBR Green PCR Core Reagents kit (Perkin-Elmer Applied
Biosystems). The nucleotide sequences of the primers used are listed in
Table 1. Each sample was
analyzed in duplicate and a calibration curve was constructed in parallel for
each analysis. The level of transcripts was normalized according to the RPLP0
gene (also known as 36B4), which encodes human acidic ribosomal phosphoprotein
P0 as an endogenous RNA control, and each sample was normalized on the basis
of its RPLP0 content.
Immunoblot analysis
Cells were washed twice with ice-cold PBS, scraped and lysed at 4°C in
a buffer containing 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl
sulphate and 50 mM Tris-HCl (pH 8) supplemented with protease inhibitors. The
lysates were incubated at 4°C for 10 minutes and then centrifuged for 10
minutes at 10,000 g to pellet the nuclei. Protein
concentration was determined according to Bradford's method (BioRad, USA)
using bovine serum albumin as the standard. Supernatants were then frozen at
70°C until further analysis. Immunobloting was performed in
accordance with standard procedures. Cell lysates (30 µg) were mixed 3:1
(vol:vol) in a 100 mM Tris HCl (pH 6.8) buffer containing 1% sodium dodecyl
sulphate (SDS), 10% glycerol, 5% ß-mercaptoethanol. They were heated at
95°C for 15 minutes and then loaded on 10% SDS-PAGE (polyacrylamide gel
electrophoresis) gels. After transfer onto nitrocellulose membranes, the
membranes were incubated in Trisbuffered saline with 5% milk powder and
0.05% Tween 20 overnight at 4°C. Immunostaining was performed in the same
buffer with 1% powdered milk. The blots were probed with the following
antibodies: a mouse monoclonal anti-connexin 43 (Transduction Laboratories) at
1/1000 dilution and a cytokeratin 7-specific monoclonal antibody (dilution
1:1000, Dako). Finally, blots were developed by using horse-radish
peroxidase-conjugated antibodies (Jackson) and an enhanced chemiluminescence
kit (Pierce supersignal, Interchim France).
Statistical tests
Statistical analysis was performed using the StatView F-4.5 software
package (Abacus Concepts, Inc., Berkeley, CA). Values are presented as
mean±s.e.m. Significant differences were identified using Mann-Whitney
analysis for hormonal secretions and ANOVA for antisense studies;
P<0.05 was considered significant.
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Results |
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|
Effects of Cx43 antisense on the morphological differentiation of
trophoblasts
In vitro, purified mononuclear cytotrophoblasts isolated from normal human
term placentas aggregate and then fuse, forming the multinucleated
syncytiotrophoblast. The fusion and differentiation of isolated human
cytotrophoblast cells have been monitored by staining cells with
anti-desmoplakin antibodies to reveal cell boundaries
(Alsat et al., 1996;
Douglas and King, 1990
).
Indeed, in our experiments the desmoplakin staining present at the
intercellular boundaries of aggregated cells progressively disappeared as the
syncytiotrophoblast is formed. After 72 hours of culture, most mononuclear
cytotrophoblasts had differentiated into syncytiotrophoblasts, as illustrated
by the gathering of numerous nuclei in a large cytoplasmic mass a
(Fig. 2a). By contrast,
cytotrophoblasts treated for 48 hours with 10 µM Cx43 antisense aggregated
but did not fuse or fused poorly. Syncytiotrophoblasts were rare, as indicated
by the persistence of desmoplakin immunostaining at the intercellular
boundaries of aggregated cells (Fig.
2b).
|
To assess further the direct involvement of Cx43 in cell fusion and syncytium formation, we estimated the number of DAPI-stained nuclei per syncytium. During the first 3 days of culture, the number of nuclei per syncytium increased (Fig. 3). For instance, after 72 hours of culture, 33% of syncytia observed contained more than 12 nuclei. By contrast, in the presence of Cx43 antisense, ST formation was impaired; only small syncytia with three to six nuclei were observed. At 72 hours, no syncytia with more than nine nuclei were observed, illustrating that Cx43 antisense disrupts cell-cell fusion.
|
Functional analysis of the effects of Cx43 antisense on cell-cell
communication
We used the gap-FRAP method to study the effect of Cx43 antisense on GJIC.
GJIC was previously analyzed between cultured contiguous trophoblastic cells
(Cronier et al., 1997),
illustrating that dye could diffuse between cytotrophoblastic cells, between
cyto- and syncytiotrophoblasts and between syncytiotrophoblasts. In our study,
the presence of scrambled antisense in the culture medium for 48 hours did not
significantly affect the trophoblastic cell-to-cell communication (5.3% of
coupled cells) compared with standard conditions (6%). By contrast, the
presence of Cx43 antisense in the culture medium dramatically reduced the
percentage of coupled trophoblastic cells
(Fig. 4).
|
Effects of Cx43 antisense on gene expression and hormonal
secretion
As previously reported, the formation of the syncytiotrophoblast by the
fusion of cytotrophoblasts is associated with significant increases in
hCGß mRNA and hCG secretion. Cells treated with Cx43 antisense contained
less hCGß mRNA and secreted less hCG into the culture medium at 48 hours
(P<0.018) and 72 hours (P<0.035) than cells treated
with the scrambled antisense (Fig.
5).
|
We recently showed that the expression of HERV-W env, which is also called
syncytin, increases during ST formation
(Frendo et al., 2003) and is
directly involved in the trophoblastic fusion process
(Blond et al., 2000
;
Frendo et al., 2003
). Thus, we
used real-time quantitative RT-PCR to determine the levels of syncytin mRNA in
cytotrophoblasts. HERV-W env mRNA levels were significantly lower (33%
decrease after 48 hours of treatment; P<0.019) in Cx43
antisense-treated cells than in control cells treated with a scrambled
antisense at 48 hours, which is the time-point at which HERV-W is maximally
expressed.
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Discussion |
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We used an in vitro model of cultured villous trophoblastic cells that has
been used to study certain aspects of the dynamic processes that occur during
villous differentiation (Kliman et al.,
1986). To rule out the possibility that villous trophoblast cells
were contaminated by other cells containing Cx43 protein and transcripts, such
as endothelial and mesenchymal cells, we added an additional purification step
with a monoclonal anti-human leukocytic antigen A, B and C. Furthermore, we
thoroughly washed cultured trophoblastic cells after cellular adherence to
eliminate syncytiotrophoblast fragments
(Cronier et al., 1997
;
Guilbert et al., 2002
).
Following this procedure, 95% of cultured cells are positive for cytokeratin 7
immunostaining (a specific marker of trophoblasts). As previously described,
syncytiotrophoblast formation is associated with significant increases in
hCG mRNA, ßhCG mRNA, leptin and PGH mRNA levels
(Frendo et al., 2000b
).
Few human cell types can fuse together and differentiate into
multinucleated syncytia. This process is involved in the formation of myotubes
(Constantin and Cronier, 2000;
Mege et al., 1994
;
Wakelam, 1985
), osteoclasts
(Ilvesaro et al., 2000
;
Zambonin Zallone et al., 1984
)
and the syncytiotrophoblast (Midgley et
al., 1963
). Although they share a common morphological
differentiation process, the three cell types that are able to differentiate
into a syncytium differ notably. Owing to its position, the
syncytiotrophoblast maintains a strong polarity with an apical microvillous
membrane both in situ and in vitro and is primarily engaged in absorption,
exchanges and endocrine functions. By contrast, myotubes do not exhibit
morphological polarity. The myoblast-myotube transition first requires the
withdrawal of myoblasts from the cell to G0, whereas only the
highly differentiated cells from the large pool of cytotrophoblastic cells in
the G0 phase actually fuse with the syncytiotrophoblast
(Huppertz et al., 1998
).
Unlike the syncytiotrophoblast, osteoclasts display strong locomotor
activity.
The cell-cell fusion process involved in syncytiotrophoblast formation is
poorly understood. One membrane event thought to be involved in fusion is the
phosphatidylserine (PS) flip. Phosphatidylserine is a phospholipid that is
normally confined to the inner layer of the plasma membrane. However, before
fusion, it translocates to the outer layer and facilitates intermembrane
fusion (PS flip). Adler et al. (Adler et
al., 1995) have shown that incubation with an anti-PS antibody
inhibits the forskolin-induced syncytial fusion of choriocarcinoma cells.
According to Huppertz and colleagues
(Huppertz et al., 1998
), this
PS flip is a consequence of the activation of an initiator caspase (e.g.
caspase 8), suggesting that the molecular machinery of early apoptosis is
involved in the fusion process.
Other studies have suggested that human endogenous retroviruses play an
important role (Blond et al.,
2000; Mi et al.,
2000
). Indeed, the production of recombinant syncytin (a
glycoprotein encoded by Env-W retrovirus) in a variety of cell types induces
the formation of giant syncytia. Furthermore, the fusion of a human
trophoblast cell line expressing endogenous syncytin is inhibited by an
antisyncytin antiserum. Syncytin is highly expressed in normal human
trophoblasts and recently we showed using the same antisense strategy that
syncytin is involved in human trophoblast cell fusion and differentiation
(Frendo et al., 2003
).
In this study, we show that Cx43 expression is also involved in cell
fusion. Gap junctions have been implicated in placental development (for a
review, see Winterhager et al.,
2000). Ultrastructural studies have detected gap junctions between
the trophoblastic layers in placentas (de
Virgiliis et al., 1980
; Firth
et al., 1980
; Malassiné
and Leiser, 1984
). Furthermore, gap junctions are present during
cytotrophoblastic cell fusion in the guinea-pig placenta
(Firth et al., 1980
). In human
trophoblast, we have previously shown that Cx26, Cx32, Cx33, Cx40 and Cx45 are
not detected, whereas Cx43 mRNA and protein are present. Furthermore, using
gap-FRAP we have shown the presence of a functional gap junctional
intertrophoblastic communication preceeding trophoblastic fusion. The low
occurrence of coupled cells observed (5.3% after 2 days of culture) argues for
a brief duration or paucity of this gap junctional communication. This is in
accordance with the fact that in trophoblast primary cultures, gap junctions
are only observed in a low number of cells with transmission electron
microscopy (Cronier et al.,
1994
).
The role of gap junctions in differentiation can be studied by chemically
inhibiting gap junctional communication, by using an antisense oligonucleotide
approach or by overexpressing connexin genes. Several lypophilic substances,
such as aliphatic alcohols, and compounds isolated from liquorice roots
(glycyrrhetinic acid) can uncouple gap junctional channels. Long-term
incubation with heptanol considerably reduces the degree of fusion of cultured
myoblasts (Constantin and Cronier,
2000; Mege et al.,
1994
) and preliminary studies showed that heptanol reversibly
decreases gap junctional intertrophoblastic communication and trophoblastic
cell fusion. Although its exact mechanism of action is not known, heptanol
seems to decrease the fluidity of membranous cholesterol-rich domains
(Johnston et al., 1980
;
Takens-Kwak et al., 1992
)
leading to a decrease of the open probability of junctional channels.
Furthermore, in cultured neonatal rat cardiomyocytes, heptanol does not
decrease the number of gap junctional channels, and in pancreatic acinar
cells, there is a cessation of GJIC, although gap junctions remain
structurally intact (Chanson et al.,
1989
). Nevertheless, heptanol have been implicated in other
biological processes. For example, it could modulate the activity of nicotinic
acetylcholine receptor channels in cultured rat myotubes
(Murrell et al., 1991
) and the
Ca2+-activated K+ channel expressed in Xenopus
oocyte (Chu and Treistman,
1997
). Furthermore, heptanol and octanol were thought to affect
some of the initiating responses of intracellular calcium elevation
(Venance et al., 1998
). The
possibility of a non-uncoupling action for heptanol and its nonspecific action
blocking all the connexin channels led us to develop an antisense strategy. It
was shown in this study that treatment with a scrambled antisense does not
affect the functional gap junctional communication and trophoblast
differentiation, whereas treatment with Cx43 antisense abolishes gap
junctional communication and reduces trophoblast differentiation. These data
showed the major implication of Cx43 expression in gap junctional
communication and cytotrophoblastic cell-cell fusion.
The main effects of Cx expression have been attributed to gap junctional
communication. The existence of cell-to-cell channels allows the exchange of
second messengers between aggregated trophoblastic cells, and this exchange
may regulate the fusion process. The nature of the messengers involved in the
intertrophoblastic gap junctional communication needs to be addressed in the
near future. It is conceivable that Ca2+, IP3 and cAMP
are exchanged, thus controlling various cellular effectors involved in fusion
and in the transcription of syncytiotrophoblast-specific genes
(Aronow et al., 2001;
Keryer et al., 1998
). These
intercellular messengers may also crosstalk with GJIC, as gap junction
channels are regulated by cAMP and Ca2+. Thus, fusion may be
correlated with a concomitant increase in cellular levels of cAMP
(Roulier et al., 1994
) and
with a decrease in basal Ca2+ activity
(Cronier et al., 1999
).
In humans, data concerning GJIC and trophoblast differentiation have been
obtained in vitro, and recently the principles of placental development have
been explained by gene knockout approaches in mice
(Rossant and Cross, 2001).
Owing to the striking diversity in placental structure and endocrine function,
we must be careful when extrapolating findings regarding placental development
from one species to another. In mice, Cx26 and Cx31 deficiencies cause
placental alterations (Gabriel et al.,
1998
; Plum et al.,
2001
), whereas in the human placenta, Cx26 and Cx31 are not
expressed.
In conclusion, cell fusion is the limiting factor in human villous
trophoblast differentiation and studies are still required to improve our
understanding of the various factors directly involved in human trophoblast
fusion and differentiation. In this study, we show for the first time that
Cx43 is one of the componants involved in these processes. Pathological
models, such as cytotrophoblasts isolated from T21-affected placentas
(Frendo et al., 2001) and in
which cell fusion and syncytiotrophoblast formation are defectuous
(Frendo et al., 2000b
), should
help to further our understanding of the cell-cell fusion process.
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Acknowledgments |
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