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Program in Development and Fetal Health, Samuel Lunenfeld Research Institute, Mount Sinai Hospital (I.C., S.J.L., J.C.C.), and Departments of Obstetrics and Gynaecology (S.J.L., J.C.C.), Pediatrics (I.C.), Physiology (S.J.L.), and Molecular and Medical Genetics (J.C.C.), University of Toronto, Toronto, Ontario, M5G 1X5, Canada
Address all correspondence and requests for reprints to: Dr. James Cross, Program in Development and Fetal Health, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5 Canada. E-mail: cross{at}mshri.on.ca
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Introduction |
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The stepwise progression starting from villous cytotrophoblasts, to transitional extravillous cytotrophoblasts in the column, finally to invasive extravillous cytotrophoblasts is characterized by dramatic changes in the expression of cell adhesion molecules and proteinases that degrade the extracellular matrix (9, 10). Understanding the importance of these changes has been assisted by the development of systems for culturing isolated cytotrophoblast cells. Invasion in vitro is dependent on the expression of MMPs, particularly MMP-9 (gelatinase B) (11). While in vitro differentiation of villous cytotrophoblast cells into either syncytiotrophoblast or invasive cytotrophoblasts can occur under relatively simple culture conditions, several factors can influence cytotrophoblast cell differentiation. For example, culturing first trimester cytotrophoblasts in lowered oxygen tension arrests their acquisition of invasiveness and sustains their proliferation (12). Cytokines and growth factors produced locally also affect cytotrophoblasts in vitro. For example, hepatocyte growth factor (13) and vascular endothelial growth factor (14) can stimulate trophoblast DNA replication. Transforming growth factor (TGF)-ß suppresses cytotrophoblast invasion (15, 16, 17) and endocrine differentiation (18), whereas IL-1 increases cell invasiveness (19). Epidermal growth factor (EGF) has multiple effects, promoting either cell proliferation (20) or invasion (21) depending on the gestational age. The influence of growth factors and cytokines on interac-tions between cell types in the intact villus, such as can be studied using villus explant cultures, have been less well documented.
Activin is also produced in the human placenta (22, 23, 24, 25, 26). The presence of activin receptors on cytotrophoblast cells suggests that activin may have autocrine or paracrine effects (27). In support of this idea, addition of activin to cultured cytotrophoblast cells stimulates their production of human CG (hCG) and progesterone (22, 28, 29). Because activin is produced by cytotrophoblast cells themselves, the response to exogenous hormone may at first seem somewhat paradoxical. This is reconciled by the fact that trophoblast cells are also a source of inhibin, a hormone related to activin that can antagonize the effects of activin on hCG secretion (28). The activin binding protein, follistatin, is also produced in the placenta (30). Like inhibin, follistatin antagonizes the effects of activin in a variety of systems, including the endocrine activity of cultured cytotrophoblasts, in which it has been shown to affect GnRH-stimulated hCG secretion from choriocarcinoma cells (30). This suggests that the activity of the activin system depends significantly on the degree to which inhibin and follistatin are also expressed.
The objective of the current study was to explore the possibility that activin and follistatin play general roles in cytotrophoblast development, apart from their endocrine effects. To do this, we cultured explants of first trimester chorionic villi which under normal circumstances retain the normal architecture of floating villi over several days. Addition of activin to these cultures, however, induced a massive outgrowth of cytotrophoblast cells that differentiated along the normal pathway including invasion into the surrounding matrigel.
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Materials and Methods |
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Chorionic villus explant cultures
Chorionic villous explant cultures were established from first
trimester human placentae as described in detail previously (17, 33, 34). Briefly, placentae from 5–7 week elective pregnancy terminations,
derived by dilatation and curettage, were dissected to remove
endometrial tissue and fetal membranes. Small fragments of villus tips
(15–20 mg wet weight) were placed on Millicell-CM culture dish inserts
(Millipore Corp, Bedford, MA) that were precoated with 0.2 ml of
undiluted matrigel (Collaborative Research, Inc., Bedford, MA) and
placed in 24-well plates. Explants were cultured in DMEM/F12 (GIBCO,
Grand Island, NY) supplemented with 100 µg/ml streptomycin, 100 U/ml
penicillin, and 0.25 mg/ml ascorbic acid, pH 7.4. In all experiments, a
single placenta was used and triplicate explants were set up for each
treatment. Explants were incubated overnight in regular medium to allow
attachment to the matrigel before the addition of treatments.
The next day, media were replaced with either regular medium (control), or medium supplemented with activin A (0.1–10 ng/ml), inhibin A (10 ng/ml), follistatin (100 ng/ml), TGF-ß3 (10 ng/ml), a monoclonal antibody reactive with human endoglin (MAb 44G4 IgG, 10 µg/ml), or antisense phosphorothioate oligonucleotides (10 µM) used to block endoglin or TGF-ß3 synthesis. The antisense oligonucleotides were directed against sequences around the translation start site (Endoglin: 5'-GCGTGCCGCGGTCCAT-3'; TGF-ß3: 5'-CCTTTGCAAGTGCATC-3'), whereas the sense sequences were used as negative controls. Culture media including treatments were routinely changed every 48 h thereafter. hCG and progesterone concentrations in conditioned media were measured by RIA (Coat-A-Count, DPC, Los Angeles, CA).
Immunostaining
Villous explants were fixed for 1 h at 4°C in 4%
(wt/vol) paraformaldehyde and then embedded in OCT compound (Tissue
Tek, Miles, IN) for cryosectioning. Ten micron sections were subjected
to immunostaining using a rat monoclonal antibody directed against
cytokeratin (used at 1:100) followed by horseradish peroxidase
conjugated antirat IgG (Sigma, St. Louis, MO; used at 1:50), and mouse
monoclonal antibodies against MHC class I (used at 1:1000), MMP-2 (used
at 1:100) and MMP-9 (used at 1:100) followed by horseradish peroxidase
conjugated antimouse IgG (Amersham, Buckinghamshire, UK; used at 1:50).
Reactions were developed using diaminobenzidine substrate (Sigma, St.
Louis, MO).
BrdU labeling
Villous explants exposed to activin (10 ng/ml) for 2 days, were
then incubated for 6 h in the presence of 1 µM
5-bromo-2'-deoxyuridine (BrdU) (Sigma, St. Louis, MO). Tissues were
fixed in Carnoy’s fixative for 1 h at 4 C, then embedded in OCT.
BrdU-positive cells were detected using biotin conjugated, anti-BrdU
(Zymed, South San Francisco, CA; used at 1:1000) and streptavidin-TRITC
(Sigma; used at 1:150). Cell nuclei were stained with bisbenzimide
(Sigma). For some experiments, streptavidin-horseradish peroxidase was
used as the secondary antibody (Sigma; used at 1:150).
Fibronectin synthesis and release
On day 5 of culture, media were replaced by methionine- and
cysteine-free, low glucose DMEM containing treatments and 25 µCi/ml
of [35S]-methionine/cysteine. After 18 h, media were
collected and diluted with an equal volume of 25 mM
Tris-HCl buffer, pH 7.4, 0.15 M NaCl and 0.5% (vol/vol)
Triton X-100, and fibronectin was isolated by binding to
gelatin-Sepharose as previously described (35). Briefly, 50 µl of a
gelatin-Sepharose suspension (Pharmacia Biotech, Uppsala, Sweden) was
added to 500 µl of diluted medium and the samples were incubated
overnight at 4 C. The gelatin-Sepharose beads were centrifuged, washed
three times in Tris/Triton X-100 buffer. Proteins were eluted by
boiling for 5 min in 1% (vol/vol) SDS and separated on a 4–12%
(wt/vol) polyacrylamide gradient gels (NOVEX, San Diego, CA).
Radioactive bands were revealed by autoradiography. Gels typically
showed a single band around 200 kDa, characteristic of fibronectin. The
radiolabeled band at 200 kDa was then quantitated using a PhosphoImager
(410A and Image Quant software, Molecular Dynamics, Sunnyvale, CA).
Detection of metalloproteinases by zymography and Western
blotting
Conditioned media were harvested from explants at day 1, 2 and 5
of treatment. Two microliters of media were mixed with 10% (vol/vol)
glycerol, 2% (wt/vol) SDS, 0.0025% (wt/vol) bromophenol blue, 0.5
M Tris, pH 6.8 and subjected to substrate-gel
electrophoresis on a 10% polyacrylamide gel (wt/vol) impregnated with
0.1% gelatin (NOVEX, San Diego, CA). After electrophoresis, gels were
washed in 2.5% (vol/vol) Triton X-100 washes (2 x 30 min at room
temperature) to remove the SDS, then equilibrated with developing
buffer (50 mM Tris-HCl, 0.2 M NaCl, 5
mM CaCl2, Brij 35, pH 7.2) for 30 min and
incubated overnight in the same buffer at 37 C. They were stained with
0.1% (wt/vol) Coomassie brilliant blue G-250 then destained to
visualize zones of gelatinase activity.
For Western blot analysis of MMP expression, 5 µl of conditioned media were electrophoresed through 10% SDS-polyacrylamide gels. Proteins were then blotted to Westran (Schleicher and Schuell, Keene, NH) PVDF membrane. Primary antibodies were used at 1:100 and detected using horse radish peroxidase conjugated antimouse IgG (Amersham; used at 1:10,000) and enhanced chemiluminescence (ECL; Amersham).
Statistical analysis
Differences among treatment means were assessed by t
test, or ANOVA and Student-Newman-Keul’s test.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
However, addition of recombinant activin-A to the culture media
stimulated substantial outgrowth of cells from a region that was
restricted to the villous tip (Fig. 1B). The effect of activin was very
rapid, being apparent as early as 12 h after addition, though
outgrowth continued over the course of several days (Fig. 1A).
Significant outgrowth was observed at doses of 1 and 10 ng/ml, but not
0.1 ng/ml (data not shown). Recombinant inhibin in the same dose range
was without effect (data not shown). Staining with anticytokeratin
confirmed that all of the cells in the villous outgrowths from
activin-treated explants were cytotrophoblasts (Fig. 2B). Because activin was previously shown
to stimulate the production of progesterone and hCG from isolated
cytotrophoblast cells in culture, we also measured the release of these
hormones by cultured explants. Cumulative production of both
progesterone and hCG increased throughout the course of the 5-day
culture period and was significantly elevated by activin treatment (see
Fig. 8; P < 0.05).
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). Counting cells
on several histological sections showed that the labeling index of
cytotrophoblast cells was highest in the villous cytotrophoblast
population and dropped progressively as cells moved distally within the
extravillous region (Table 1). Although
we have not addressed this point, we cannot rule out the possibility
that activin may first induce higher levels of cell division in the
villous-associated cytotrophoblast population, an effect secondarily
leading to more extravillous cells. Nonetheless, these data indicate
that cells in the cytotrophoblast outgrowth are primarily not cycling,
and therefore their appearance in the outgrowth must have been due to
the migration of cells away from the villous core. Within the
outgrowths, epithelial cell morphology changed dramatically.
Cytotrophoblast cells close to the stroma were smaller and tightly
clustered, whereas more distal cells became progressively larger and at
the outgrowth edge were often isolated in the surrounding matrigel
(Fig. 2B). These morphological features were reminiscent of the
cytotrophoblast cell phenotypes within anchoring type chorionic villi
in vivo.
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5ß1 integrin (9) as well as express
its ligand, fibronectin (36). The synthesis of fibronectin was 3- to
8-fold higher in activin-treated explants compared with controls (Fig. 3), consistent with the presence of
significantly more transitional cytotrophoblast cells. Human leukocyte
antigen (HLA)-G is expressed on the surface of extravillous
cytotrophoblast cells in the distal portion of the column and in
invasive cells that enter the decidua (37, 38, 39). Immunostaining of
activin-treated explants with monoclonal antibody W6/32, which
recognizes HLA-G, demonstrated expression on cells in the distal
portions of the outgrowth and in cells that were invading the
surrounding matrigel (Fig. 2F).
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).
MMP-9 is produced as a 92-kDa proenzyme, which becomes activated by
cleavage to form an 84-kDa species. It is likely that the gelatinase
activity at 84 kDa was due to activated MMP-9 because Western blotting
with an MMP-9-specific monoclonal antibody identified an immunoreactive
band at 84 kDa (Fig. 4B). The relatively weak 84-kDa immunoreactive
band observed was consistent with the observation on the zymogram that
the comigrating gelatinolytic activity at 84 kDa appeared to be of
relatively lower abundance. Because MMP-9 is known to be produced by
activated macrophages, which might have been present in the stroma of
the cultured villi, we performed immunostaining to determine if it was
expressed by cytotrophoblast cells. The MMP-9 monoclonal antibody
detected scattered, weakly positive cells in the villous stroma (Fig. 2G). MMP-9-positive (albeit weakly) cytotrophoblast cells were
also present but were largely restricted to the distal region
of the outgrowth and in cells invading the matrigel in control explants
(Fig. 2G). This restricted site of expression was similar to the
pattern of HLA-G staining (Fig. 2F).
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). There was also no
difference in the extent to which MMP-9 was activated. Immunostaining
of activin treated explants showed that activin treatment also did not
apparently affect the site of MMP-9 expression (data not shown).
Activin increases MMP-2 production by villous cytotrophoblast
cells
Besides MMP-9, zymography showed that villous explants also
released large amounts of gelatinase activity at 68 and 60 kDa, which
are the sizes of the proenzyme and activated forms of MMP-2,
respectively (Fig. 4A). The lower molecular weight bands around 60 kDa
may also represent interstitial collagenase, which is known to be
produced by isolated cytotrophoblasts in culture (40). The release of
both these species was significantly increased by activin treatment.
Western blotting with an MMP-2-specific monoclonal antibody showed
immunoreactive proteins at 68, 60, and 32 that were abundant in the
conditioned media from activin-treated cultures, but were weak or
undetectable in control media (Fig. 4B). The identity of the 32 kDa
immunoreactive band is unknown, though it likely represents a
proteolytic fragment of MMP-2. Activin induction of MMP-2 release was
apparent even after only 1 day of treatment. Therefore, the induction
of MMP-2 was a specific and early effect of activin treatment.
To identify the cellular source of MMP-2, control explants as well as
those treated with activin for 48 h were fixed and sectioned for
immunostaining. In control explants, MMP-2 immunoreactivity was only
weakly detectable in villous stroma and extravillous cytotrophoblasts
(Fig. 2H). Activin treatment had no effect on these sites of expression
(data not shown). However, in villous cytotrophoblast cells that
underlie the syncytiotrophoblast layer, whereas MMP-2-immunoreactivity
was undetectable in control explants (Fig. 5A), it was significantly elevated in
activin-treated explants (Fig. 5B). MMP-2 immunoreactivity was also
slightly elevated in the syncytiotrophoblast of activin-treated
explants compared with controls (Fig. 5, B vs. A, data not
shown), though this effect was much less dramatic than the change in
villous cytotrophoblast expression. Together these data demonstrate
that the increase in MMP-2 expression following activin treatment is
primarily due to MMP-2 production from villous cytotrophoblast
cells.
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) and
fibronectin synthesis (Fig. 6B) compared with controls, though the
latter effect was not statistically significant in all experiments. As
noted earlier, though, the outgrowth of cytotrophoblasts from intact
villi is relatively small under control conditions, and therefore our
ability to observe a reduction was likely limited. To get around this
problem, we tested whether follistatin could block cytotrophoblast
outgrowth after it had been initiated by treatment for a short period
with activin alone. Follistatin (100 ng/ml) was therefore added after
24 h exposure to activin (10 ng/ml). The high concentration of
follistatin added was such that we were trying to block the effects of
both exogenous as well as any endogenously produced activin.
Follistatin effectively reversed the effects of exogenous activin
including morphological outgrowth (Fig. 6A) and fibronectin synthesis
(Fig. 6B). These data suggest that sustained activin function,
including via endogenously produced hormone, is required to elicit full
outgrowth.
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and 7). TGF-ß isoforms are expressed by
various cells in villous explants, and the fact that inhibition of
their expression or their signaling, by interference with receptor
expression, indicates that TGF-ß functions as a negative regulator of
cytotrophoblast outgrowth. To determine if exogenous TGF-ß could
block activin-stimulated outgrowth, we added TGF-ß3 (10 ng/ml) to
explants treated initially for 24 h with activin (10 ng/ml). Under
these conditions, the effects of TGF-ß3 were similar to those of
follistatin in suppression of cytotrophoblast outgrowth (Fig. 6A).
Interestingly, however, TGF-ß3 did not significantly reduce the
activin-stimulated increase in fibronectin synthesis (Fig. 6B).
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). TGF-ß signaling
was blocked in two ways; ligand expression was reduced using antisense
oligonucleotides designed against TGF-ß3 (AS-ß3) (Fig. 6) and
receptor function was affected through inhibition of endoglin, a
TGF-ß1 and -ß3 binding protein, with a monoclonal antibody (Fig. 7). While both methods elicited outgrowth, as has been observed before
(17), these effects were inhibited by follistatin. This effect was most
significant with simultaneous addition of follistatin and the outgrowth
inducer. However, when addition of follistatin was delayed for 24
h, it was noteworthy that although outgrowth was significantly reduced
by follistatin, morphological outgrowth was still significantly greater
compared with control or follistatin alone treatment (Fig. 6A).
The effect of follistatin on biochemical markers of cytotrophoblast
differentiation was also examined. Like the effect of activin,
inhibition of TGF-ß signaling resulted in increased production of
fibronectin (Figs. 6B, 7A), gelatinase activity at 68 and 60 kDa (Fig. 7B), hCG (Fig. 8), and progesterone (Fig. 8). All of these effects were reversed to control levels by addition of
follistatin to the cultures (
Figs. 6–8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Activin induces cytotrophoblast outgrowth
The cytotrophoblast cell outgrowths induced by activin resemble by
morphology, sites of cell proliferation and expression of specific
markers, those cytotrophoblast populations present in anchoring
chorionic villi. Therefore, we suppose that activin initiates an early
event leading to subsequent differentiation steps that progress
uninterrupted. That this full differentiation program could occur
in vitro is supported by the fact that purified
cytotrophoblast cells undergo the same process (40, 41, 42, 43). Therefore, the
villous explant system is unique in that unless an initiating event
occurs to trigger the process, villous cytotrophoblast cells within the
explant remain relatively static (33, 34).
The mechanism by which activin induces cytotrophoblast outgrowth is obscure at present. Though activin and follistatin have effects in a variety of systems (44), effects on trophoblast cell differentiation or migration, as described here, appear unique. The closest parallel may be the effect of activin on promoting mesoderm differentiation, a process best studied in Xenopus embryos. During gastrulation, a specialized region of embryonic ectoderm transforms into mesoderm involving extensive cell migration and presumed changes in cell-cell or cell-extracellular matrix interactions (45, 46). Indeed, activin induces changes in integrin expression on Xenopus ectodermal cells (45). In a similar manner, during the transition from villous to extravillous cytotrophoblast cells, cells must first detach from the villus basement membrane, then migrate through the cytotrophoblast column and ultimately invade the decidua. Each step in this process in characterized by the expression of distinct sets of integrins (9) and cell-cell adhesion molecules (e.g. cadherins) (40). Additionally, cytotrophoblast cells express extracellular matrix-degrading enzymes such as plasminogen activator (41, 42) and metalloproteinases (11, 47) that are thought to be required for migration and invasion. MMP-9 is required for at least the terminal step of cytotrophoblast differentiation, invasion into the laminin-rich decidua, a process that can be studied by invasion of matrigel in vitro (11). Our direct demonstration here by immunostaining that MMP-9 is expressed by cells only at the distal fringes of cytotrophoblast outgrowths in culture supports this hypothesis and is similar to the expression pattern of MMP-9 in vivo (48).
Activin stimulates MMP-2 production by villous cytotrophoblast
cells
A significant early effect of activin on the villous explants was
the induction of MMP-2 expression. The 60-kDa form of MMP-2 was
stimulated by activin, as well as the 68-kDa proenzyme form, indicating
that the enzyme becomes activated. The role of MMP-2 in cytotrophoblast
differentiation has been somewhat controversial. By zymography
analysis, MMP-2 is abundantly expressed by purified cytotrophoblast
cells using some procedures (47), whereas in others it is barely
detectable (11). The discrepancy has been attributed to the possibility
of placental stromal cells contaminating the cultures. The fibroblasts
could be a source of MMP-2 directly or may redirect cytotrophoblast
differentiation toward syncytiotrophoblast (49), also a potential
source of MMP-2. Our data support a third and more interesting
possibility. The fact that MMP-2 expression by villous cytotrophoblast
cells was significantly elevated in activin-treated explants at a time
when extensive transition toward extravillous outgrowth was occurring,
argues that activated villous cytotrophoblast cells are a significant
source of MMP-2. They presumably would use this enzyme to migrate off
the basement membrane. Thereafter, in the cytotrophoblast column,
expression of MMP-2 is significantly reduced. Given this, the extent to
which MMP-2 is expressed by freshly isolated cytotrophoblast cells may
depend significantly on the proportion of cells in the population that
represent the earliest stages of differentiation. The fact that freshly
isolated cytotrophoblasts often contain large fractions of 5 and
even 1 integrin-positive cells (9, 32, 36), which represent
extravillous populations, strongly argues that the numbers of these
early cells may be low. This may be particularly true if older
placentae are used for cell isolation because cytotrophoblast
proliferation and invasiveness changes dramatically within the first
trimester. An advantage of the explant system is, therefore, the
ability to better study the behavior of villous cytotrophoblast cells
in culture.
We conclude that activin plays an autocrine or paracrine role in modulating cytotrophoblast differentiation within chorionic villi. In both first and third trimester villi, activin expression has been detected in both syncytiotrophoblasts and the underlying cytotrophoblasts (24, 25, 26). In contrast, follistatin is expressed by syncytiotrophoblasts (30). Because receptors for activin are present on cytotrophoblasts (27), activin can have autocrine and paracrine effects in intact villi. Explants of floating chorionic villi placed into culture essentially consist only of a villous cytotrophoblast monolayer with covering syncytiotrophoblast, and an underlying basement membrane and stromal core. We suggest that a likely target for the effects of activin is the villous cytotrophoblast cell whose production of MMP-2 is acutely increased in response to activin. At the moment we cannot rule out the possibility that activin may play additional roles at subsequent steps along the progression ultimately toward invasive cytotrophoblasts. This would be best tested using isolated cytotrophoblast cells which, when put into culture, represent an intermediate cell population partially progressed along the differentiation pathway.
Activin and TGF-ß have opposing effects on cytotrophoblast
differentiation
Recently, TGF-ß and activin were shown to have opposing effects
on hCG production by human trophoblast cells (50), but the mechanisms
of their effects were unexplored. In our studies, the discovery that
activin has effects on villus development that are opposite to that of
TGF-ß, and moreover that cytotrophoblast outgrowth induced by
inhibiting TGF-ß signaling is reversed in turn by follistatin (an
inhibitor of activin), indicates that the effects of these growth
factors intersect to regulate cytotrophoblast differentiation. Though
it is not surprising that growth factor networks affect complex
biological events, this is the first study to systematically determine
at what steps in cytotrophoblast differentiation different growth
factors act and how their effects are integrated. The ability to induce
the activin and TGF-ß signaling pathways by the addition of ligands,
as well as to block the effects with follistatin and antiendoglin,
respectively, or with antisense oligonucleotides will allow the
interactions between these cytokines to be described in further detail.
Other growth factors and cytokines have been shown to affect
cytotrophoblast growth and differentiation, including IL-1 (19),
epidermal growth factor (20, 21), hepatocyte growth factor (13), and
vascular endothelial growth factor (14). Because it is likely
that these factors function as a network, it would be fruitful to use
inducing and blocking strategies similar to the ones used here to
unravel this complexity. Such strategies will likely be necessary to
assign these different factors to regulating specific steps in
cytotrophoblast differentiation. This critical understanding will be
important to evaluate what specific processes are affected in a disease
such as preeclampsia in which villous development and cytotrophoblast
invasion are abnormal.
In considering how activin and TGF-ß pathways may intersect to
regulate cytotrophoblast development, we consider two general
possibilities. In the first, activin and TGF-ß independently regulate
processes that control the ability of cytotrophoblast cells to migrate
and differentiate along the invasive pathway. The sum of the inhibitory
effects of TGF-ß, the inducing effects of activin and blocking
effects of its inhibitor, follistatin, would determine whether a
floating-type villus tip develops sprouts of cytotrophoblasts to form
an anchoring type villus. Alternatively, TGF-ß and activin could act
in series. TGF-ß, which could be produced by syncytiotrophoblast
(51), would act on the villous cytotrophoblasts and reduce activin
expression or activity (perhaps through follistatin production or
effects on activin receptor expression). Activin mediated processes
necessary for cytotrophoblast migration and differentiation would in
turn therefore be reduced. Based on our current data, we suggest that
elements of both pathways may be correct and propose a general model
summarized in Fig. 9. The basis of the
model is the finding that, whereas exogenous TGF-ß3 was a potent
inhibitor of cytotrophoblast outgrowth, it was unable to block
activin-induced increase in fibronectin synthesis. Conversely,
follistatin ablated fibronectin synthesis, but only effectively blocked
morphological outgrowth when added at the start of the experiment.
Together these data suggest that while activin and TGF-ß may each
have direct effects on cytotrophoblast cells, activin may be the direct
regulator of certain responses, such as fibronectin synthesis, and
TGF-ß effects may be indirect. Other experimental approaches that may
be useful to clarify this pathway include determining if the effects of
inhibition of TGF-ß action and addition of activin have additive
effects; if all effects of TGF-ß occur upstream of activin one would
predict that effects would not be additive. Such actions of TGF-ß
could be exerted through direct effects on activin production, or
through induction of either follistatin, the activin inhibitor, or
inhibin, a factor which has opposing effects in other cell systems,
such as luteal cells (52). Conversely, TGF-ß could affect activin
receptor expression or downstream signaling events. Ongoing experiments
will explore the nature of the interaction between activin and TGF-ß
in detail, by testing some of these hypotheses.
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Acknowledgments |
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Footnotes |
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2 Career Scientist of the Ontario Ministry of Health.
3 Medical Research Council of Canada Scholar.
Received April 14, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M.C. Ferreira, C.A. Witz, L.S. Hammes, N. Kirma, F. Petraglia, R.S. Schenken, and F.M. Reis Activin A increases invasiveness of endometrial cells in an in vitro model of human peritoneum Mol. Hum. Reprod., May 1, 2008; 14(5): 301 - 307. [Abstract] [Full Text] [PDF]
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 W. V. Ingman and R. L. Jones Cytokine knockouts in reproduction: the use of gene ablation to dissect roles of cytokines in reproductive biology Hum. Reprod. Update, March 1, 2008; 14(2): 179 - 192. [Abstract] [Full Text] [PDF]
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 K. Y Arai and T. Nishiyama Developmental Changes in Extracellular Matrix Messenger RNAs in the Mouse Placenta During the Second Half of Pregnancy: Possible Factors Involved in the Regulation of Placental Extracellular Matrix Expression Biol Reprod, December 1, 2007; 77(6): 923 - 933. [Abstract] [Full Text] [PDF]
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 N. Antony, J. J. Bass, C. D. McMahon, and M. D. Mitchell Myostatin regulates glucose uptake in BeWo cells Am J Physiol Endocrinol Metab, November 1, 2007; 293(5): E1296 - E1302. [Abstract] [Full Text] [PDF]
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M. Myers, E. Gay, A. S. McNeilly, H. M. Fraser, and W. C. Duncan In Vitro Evidence Suggests Activin-A May Promote Tissue Remodeling Associated with Human Luteolysis Endocrinology, August 1, 2007; 148(8): 3730 - 3739. [Abstract] [Full Text] [PDF]
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 G. St. J. Whitley, P. R. Dash, L.-J. Ayling, F. Prefumo, B. Thilaganathan, and J. E. Cartwright Increased Apoptosis in First Trimester Extravillous Trophoblasts from Pregnancies at Higher Risk of Developing Preeclampsia Am. J. Pathol., June 1, 2007; 170(6): 1903 - 1909. [Abstract] [Full Text] [PDF]
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 P. Florio, F. M. Severi, C. Bocchi, S. Luisi, M. Mazzini, S. Danero, M. Torricelli, and F. Petraglia Single Serum Activin A Testing to Predict Ectopic Pregnancy J. Clin. Endocrinol. Metab., May 1, 2007; 92(5): 1748 - 1753. [Abstract] [Full Text] [PDF]
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C. Ferretti, L. Bruni, V. Dangles-Marie, A.P. Pecking, and D. Bellet Molecular circuits shared by placental and cancer cells, and their implications in the proliferative, invasive and migratory capacities of trophoblasts Hum. Reprod. Update, March 1, 2007; 13(2): 121 - 141. [Abstract] [Full Text] [PDF]
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P. Florio, M. Rossi, P. Vigano, S. Luisi, M. Torricelli, P. B. Torres, A. M. Di Blasio, and F. Petraglia Interleukin 1{beta} and Progesterone Stimulate Activin A Expression and Secretion From Cultured Human Endometrial Stromal Cells Reproductive Sciences, January 1, 2007; 14(1): 29 - 36. [Abstract] [PDF]
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 R. L Jones, T. J Kaitu'u-Lino, G. Nie, L G. Sanchez-Partida, J. K Findlay, and L. A Salamonsen Complex expression patterns support potential roles for maternally derived activins in the establishment of pregnancy in mouse. Reproduction, November 1, 2006; 132(5): 799 - 810. [Abstract] [Full Text] [PDF]
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 J. L. James, P.R. Stone, and L.W. Chamley The effects of oxygen concentration and gestational age on extravillous trophoblast outgrowth in a human first trimester villous explant model Hum. Reprod., October 1, 2006; 21(10): 2699 - 2705. [Abstract] [Full Text] [PDF]
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R. L Jones, C. Stoikos, J. K Findlay, and L. A Salamonsen TGF-{beta} superfamily expression and actions in the endometrium and placenta. Reproduction, August 1, 2006; 132(2): 217 - 232. [Abstract] [Full Text] [PDF]
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E.K. Moses, E. Fitzpatrick, K.A. Freed, T.D. Dyer, S. Forrest, K. Elliott, M.P. Johnson, J. Blangero, and S.P. Brennecke Objective prioritization of positional candidate genes at a quantitative trait locus for pre-eclampsia on 2q22 Mol. Hum. Reprod., August 1, 2006; 12(8): 505 - 512. [Abstract] [Full Text] [PDF]
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 Y.-G. Chen, Q. Wang, S.-L. Lin, C. D. Chang, J. Chung, and S.-Y. Ying Activin Signaling and Its Role in Regulation of Cell Proliferation, Apoptosis, and Carcinogenesis. Experimental Biology and Medicine, May 1, 2006; 231(5): 534 - 544. [Abstract] [Full Text] [PDF]
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R. L. Jones, J. K. Findlay, P. G. Farnworth, D. M. Robertson, E. Wallace, and L. A. Salamonsen Activin A and Inhibin A Differentially Regulate Human Uterine Matrix Metalloproteinases: Potential Interactions during Decidualization and Trophoblast Invasion Endocrinology, February 1, 2006; 147(2): 724 - 732. [Abstract] [Full Text] [PDF]
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E. Dimitriadis, C.A. White, R.L. Jones, and L.A. Salamonsen Cytokines, chemokines and growth factors in endometrium related to implantation Hum. Reprod. Update, November 1, 2005; 11(6): 613 - 630. [Abstract] [Full Text] [PDF]
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C. Nicola, A. V. Timoshenko, S. J. Dixon, P. K. Lala, and C. Chakraborty EP1 Receptor-Mediated Migration of the First Trimester Human Extravillous Trophoblast: The Role of Intracellular Calcium and Calpain J. Clin. Endocrinol. Metab., August 1, 2005; 90(8): 4736 - 4746. [Abstract] [Full Text] [PDF]
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 C Bearfield, E Jauniaux, N Groome, I L Sargent, and S Muttukrishna The secretion and effect of inhibin A, activin A and follistatin on first-trimester trophoblasts in vitro Eur. J. Endocrinol., June 1, 2005; 152(6): 909 - 916. [Abstract] [Full Text] [PDF]
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M.-C. Lacroix, J. Guibourdenche, T. Fournier, I. Laurendeau, A. Igout, V. Goffin, J. Pantel, V. Tsatsaris, and D. Evain-Brion Stimulation of Human Trophoblast Invasion by Placental Growth Hormone Endocrinology, May 1, 2005; 146(5): 2434 - 2444. [Abstract] [Full Text] [PDF]
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 M. D. Steller, T. J. Shaw, B. C. Vanderhyden, and J.-F. Ethier Inhibin Resistance Is Associated with Aggressive Tumorigenicity of Ovarian Cancer Cells Mol. Cancer Res., January 1, 2005; 3(1): 50 - 61. [Abstract] [Full Text] [PDF]
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 A. De Luca, M. De Falco, V. Fedele, L. Cobellis, A. Mastrogiacomo, V. Laforgia, I. L. Tuduce, M. Campioni, D. Giraldi, M. G. Paggi, et al. The Serine Protease HtrA1 Is Upregulated in the Human Placenta During Pregnancy J. Histochem. Cytochem., July 1, 2004; 52(7): 885 - 892. [Abstract] [Full Text] [PDF]
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 S. Koide, I. Yoshida, A. Tsuji, and Y. Matsuda The Expression of Proprotein Convertase PACE4 Is Highly Regulated by Hash-2 in Placenta: Possible Role of Placenta-Specific Basic Helix-Loop-Helix Transcription Factor, Human Achaete-Scute Homologue-2 J. Biochem., September 1, 2003; 134(3): 433 - 440. [Abstract] [Full Text] [PDF]
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P. Florio, F. M. Severi, S. Luisi, P. Ciarmela, G. Calonaci, L. Cobellis, and F. Petraglia Endometrial Expression and Secretion of Activin A, But Not Follistatin, Increase in the Secretory Phase of the Menstrual Cycle Reproductive Sciences, May 1, 2003; 10(4): 237 - 243. [Abstract] [PDF]
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H. J. Roberts, S. Hu, Q. Qiu, P. C.K. Leung, I. Caniggia, A. Gruslin, B. Tsang, and C. Peng Identification of Novel Isoforms of Activin Receptor-Like Kinase 7 (ALK7) Generated by Alternative Splicing and Expression of ALK7 and Its Ligand, Nodal, in Human Placenta Biol Reprod, May 1, 2003; 68(5): 1719 - 1726. [Abstract] [Full Text] [PDF]
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K. Y. Arai, K. Tsuchida, K. Uehara, K. Taya, and H. Sugino Characterization of Rat Follistatin-Related Gene: Effects of Estrous Cycle Stage and Pregnancy on Its Messenger RNA Expression in Rat Reproductive Tissues Biol Reprod, January 1, 2003; 68(1): 199 - 206. [Abstract] [Full Text] [PDF]
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S. Muttukrishna, E. Jauniaux, N. Greenwold, H. McGarrigle, S. Jivraj, S. Carter, S. Elgaddal, N. Groome, and L. Regan Circulating levels of inhibin A, activin A and follistatin in missed and recurrent miscarriages Hum. Reprod., December 1, 2002; 17(12): 3072 - 3078. [Abstract] [Full Text] [PDF]
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F. M. Reis, D. D'Antona, and F. Petraglia Predictive Value of Hormone Measurements in Maternal and Fetal Complications of Pregnancy Endocr. Rev., April 1, 2002; 23(2): 230 - 257. [Abstract] [Full Text] [PDF]
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S. Luo, H. Yu, D. Wu, and C. Peng Transforming growth factor-{beta}1 inhibits steroidogenesis in human trophoblast cells Mol. Hum. Reprod., April 1, 2002; 8(4): 318 - 325. [Abstract] [Full Text] [PDF]
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R. L. Jones, L. A. Salamonsen, Y. C. Zhao, J.-F. Ethier, A. E. Drummond, and J. K. Findlay Expression of activin receptors, follistatin and betaglycan by human endometrial stromal cells; consistent with a role for activins during decidualization Mol. Hum. Reprod., April 1, 2002; 8(4): 363 - 374. [Abstract] [Full Text] [PDF]
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A. G. Moore, D. A. Brown, W. D. Fairlie, A. R. Bauskin, P. K. Brown, M. L. C. Munier, P. K. Russell, L. A. Salamonsen, E. M. Wallace, and S. N. Breit The Transforming Growth Factor-{beta} Superfamily Cytokine Macrophage Inhibitory Cytokine-1 Is Present in High Concentrations in the Serum of Pregnant Women J. Clin. Endocrinol. Metab., December 1, 2000; 85(12): 4781 - 4788. [Abstract] [Full Text]
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R.L. Jones, L.A. Salamonsen, H.O.D. Critchley, P.A.W. Rogers, B. Affandi, and J.K. Findlay Inhibin and activin subunits are differentially expressed in endometrial cells and leukocytes during the menstrual cycle, in early pregnancy and in women using progestin-only contraception Mol. Hum. Reprod., December 1, 2000; 6(12): 1107 - 1117. [Abstract] [Full Text] [PDF]
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 K. Ogawa, M. Funaba, L. S. Mathews, and T. Mizutani Activin A Stimulates Type IV Collagenase (Matrix Metalloproteinase-2) Production in Mouse Peritoneal Macrophages J. Immunol., September 15, 2000; 165(6): 2997 - 3003. [Abstract] [Full Text] [PDF]
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J. A. Keelan, R. L. Zhou, L. W. Evans, N. P. Groome, and M. D. Mitchell Regulation of Activin A, Inhibin A, and Follistatin Production in Human Amnion and Choriodecidual Explants by Inflammatory Mediators Reproductive Sciences, September 1, 2000; 7(5): 291 - 296. [Abstract] [PDF]
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F. Debieve, S. Pampfer, and K. Thomas Inhibin and activin production and subunit expression in human placental cells cultured in vitro Mol. Hum. Reprod., August 1, 2000; 6(8): 743 - 749. [Abstract] [Full Text] [PDF]
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S. Muttukrishna, R.A. North, J. Morris, J.-C. Schellenberg, R.S. Taylor, J. Asselin, W. Ledger, N. Groome, and C.W.G. Redman Serum inhibin A and activin A are elevated prior to the onset of pre-eclampsia Hum. Reprod., July 1, 2000; 15(7): 1640 - 1645. [Abstract] [Full Text] [PDF]
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J. Copeman, R. N.N. Han, I. Caniggia, M. McMaster, S. J. Fisher, and J. C. Cross Posttranscriptional Regulation of Human Leukocyte Antigen G During Human Extravillous Cytotrophoblast Differentiation Biol Reprod, June 1, 2000; 62(6): 1543 - 1550. [Abstract] [Full Text]
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 A. C. Staff, T. Ranheim, T. Henriksen, and B. Halvorsen 8-Iso-Prostaglandin F2{alpha} Reduces Trophoblast Invasion and Matrix Metalloproteinase Activity Hypertension, June 1, 2000; 35(6): 1307 - 1313. [Abstract] [Full Text] [PDF]
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X. Ni, S. Luo, T. Minegishi, and C. Peng Activin A in JEG-3 Cells: Potential Role as an Autocrine Regulator of Steroidogenesis in Humans Biol Reprod, May 1, 2000; 62(5): 1224 - 1230. [Abstract] [Full Text]
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J. D. Aplin, T. Haigh, C. J.P. Jones, H. J. Church, and L. Vicovac Development of Cytotrophoblast Columns from Explanted First-Trimester Human Placental Villi: Role of Fibronectin and Integrin {alpha}5ß1 Biol Reprod, April 1, 1999; 60(4): 828 - 838. [Abstract] [Full Text]
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 N. P. Barbara, J. L. Wrana, and M. Letarte Endoglin Is an Accessory Protein That Interacts with the Signaling Receptor Complex of Multiple Members of the Transforming Growth Factor-beta Superfamily J. Biol. Chem., January 8, 1999; 274(2): 584 - 594. [Abstract] [Full Text] [PDF]
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E. Y. Wang, L. B. Draper, E. Lee, A. Polak, P. Sluss, J. Weiss, and T. K. Woodruff Identification of Naturally Occurring Follistatin Complexes in Human Biological Fluids Biol Reprod, January 1, 1999; 60(1): 8 - 13. [Abstract] [Full Text]
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