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National Center for Infertility Research, Reproductive Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Alan L. Schneyer, Reproductive Endocrine Unit, Massachusetts General Hospital, Bartlett Hall Extension 5, Fruit Street, Boston, Massachusetts 02114. E-mail: schneyer.alan{at}MGH.Harvard.edu
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Abstract |
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In summary, as opposed to exogenous activin, endogenous activin decreased proliferation of PA1 cells even in the presence of cell surface associated FS. These results are consistent with a model in which FS acts as a barrier for exogenous (endocrine-paracrine) but not for endogenous (autocrine) activin. In addition, the higher PA1 cell responsiveness to endogenous compared with exogenous activin, suggests that activin overexpression in PA1 cells may up-regulate an activin signaling component, or down-regulate an activin signaling inhibitor.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and a ß-subunit (1). Both factors
were initially isolated from follicular fluid and identified based on
their ability to stimulate or suppress, respectively, the secretion of
FSH from cultured rat anterior pituitary cells (1). Activins have also
been shown to modulate cell differentiation and proliferation in a wide
range of tissues and cell types (2, 3, 4). They signal by interacting with
two structurally related receptors designated Type I and II, each
represented by two subtypes (ActRIA/ActRIB and ActRIIA/ActRIIB
respectively), all of which belonging to the serine/threonine kinase
receptor family (5). Inhibins are thought to antagonize activin
functions by competing for the activin Type II receptor (6, 7).
However, recent evidence suggests that inhibins might have a specific
signal transduction pathway as well (8, 9). FS is a structurally unrelated protein (10) that binds to activin in a largely nondissociable manner, resulting in biologically inactive complexes (11, 12). Alternative splicing of the sixth exon in the FS gene generates two different messenger RNAs that encode for two proteins of 288 and 315 amino acids (10). Additional naturally occurring forms of FS have been purified from porcine follicular fluid resulting from posttranslational glycosylation and/or proteolytic processing of FS 315 to FS 303 (13). FS 288 and FS 315 display similar affinity for activin (13). However, in contrast to FS 315, FS 288 can bind with high affinity to cell-surface heparan sulfate proteoglycans (13, 14). FS 315 is likely to be the major form in the serum, whereas shorter forms of FS are predominant in follicular fluid (15). FS is also expressed in a variety of tissues which largely overlap autocrine or paracrine targets for activin. Several lines of evidence indicate that FS acts as a local regulator of activin bioavailability by neutralizing the actions of activin (16, 17, 18). In this regard, FS 288 has been shown to prevent activin from binding to its receptor (19). More recently, FS 288 was shown to accelerate the uptake of activin A into pituitary cells leading to increased degradation by lysosomal enzymes, an action that might be important for activin clearance (20).
Previous studies from our laboratory have shown that the PA1 cell line, originally derived from a human ovarian teratocarcinoma, secretes large amounts of FS (21). Moreover, while exogenous activin had no effect on the proliferation of these cells, it inhibited proliferation when cells were first treated with heparin to remove cell-associated FS (21). Taken together, these observations suggest that cell surface associated FS might be acting as a barrier preventing exogenous activin from accessing its receptor. For the purpose of this study, we use the term exogenous for activin added to PA-1 cells in culture, or for its in vivo equivalent, endocrine/paracrine-derived activin. On the other hand, endogenous activin refers to activin made by transfected PA-1 cells, which is equivalent to autocrine-derived activin in vivo. We hypothesized that cell surface associated FS would differentially regulate the bioavailability of exogenous and endogenous activin by acting as a selective barrier for exogenous, but not endogenous activin. To address this question, we evaluated the action of exogenous and endogenous activin on PA1 cells. Our results suggest that in PA1 cells, cell surface associated FS selectively inhibits exogenous (endocrine or paracrine) but not endogenous (autocrine) actions of activin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Establishment of the stable activin-secreting clones
PA1 cells were transfected with a human inhibin/activin
ßA-subunit cDNA (provided by Genentech, Inc., South San
Francisco, CA) together with PSV2neo vector (CLONTECH Laboratories, Inc., Palo Alto, CA) using lipofectamine
(Gibco BRL, Grand Island, NY) according to the
manufacturer’s instructions. Twenty-four hours after transfection, 600
µg/ml of G418 sulfate (Mediatech, Herndon, VA) was added to the
culture medium. After 4 weeks of selection, positive clones were
isolated and individually plated in 24-well plates. Conditioned media
were assayed for activin A 1 week later.
[125I]Activin binding assay
Activin-A was iodinated to a specific activity of 30–35
µCi/µg using lactoperoxidase and purified by PAGE as previously
described (12). PA1 cells were plated in 6-well plates at 4 x
105 cells/well in 2 ml RPMI supplemented with 10% FBS.
After 48 h, cells were washed once with PBS and the medium was
replaced by 1 ml of binding buffer (RPMI with 0.1% BSA). Cells were
further incubated for 4 h at room temperature with
[125I]activin-A (105-1.5 x
105 cpm per ml), either with no other treatment, or in the
presence of 300 ng/ml unlabeled activin-A (from 20-fold concentrated
medium as previously described (22)), 25 µg/ml heparan sulfate
(Sigma Chemical Co., St. Louis, MO), 25 ng/ml rhFS 288
(National Hormone Pituitary Program, NICHD), or 25 ng/ml rhFS 288
together with 25 µg/ml heparan sulfate. Cells were then washed once
with PBS and solubilized in 1 ml of 0.5 N sodium hydroxide.
The cell-bound radioactivity was counted in a 
counter.
Proliferation experiments
Cell proliferation was quantified by [3H]thymidine
incorporation. Transfected clones and untransfected PA1 cells were
plated in 24-well plates at 2.5 x 104 cells/well in 1
ml RPMI supplemented with 10% FBS. After 24, 48, 72, and 96 h of
culture, [3H]thymidine (1.0 µCi/ml) in 10 µl medium
was added to different sets of quadruplicate wells and incubation was
continued for another 4 h. For each clone, free FS and total
activin-A were measured at 96 h of culture in the conditioned
medium of an identical well. To determine [3H]thymidine
incorporation, cells were washed once with RPMI and precipitated with
0.5 ml 10% cold trichloroacetic acid for 20 min at 4 C. The
precipitate was washed with methanol, solubilized in 0.5 ml 1
N NaOH and neutralized with 0.5 ml 1 N HCl. The
amount of radioactivity was counted in a scintillation counter (Tm
Analytic, Elk Grove Village, IL).
Two-site solid-phase immuno-chemiluminescent assay (SPICA) for
human follistatin
FS was measured in a two-site SPICA assay using two monoclonal
antibodies to nonoverlapping epitopes as previously described (23). The
intra and interassay coefficients of variation were between 2.7–4.9%
and 7.8–11.7% respectively. The assay detection limit was 1 ng/ml. No
cross-reactivity was observed with inhibin-A, activin-A, or
2 macroglobulin (23). This assay only detects free,
nonactivin bound FS (23).
Activin A ELISA
Total activin-A was measured in a two-site solid-phase
commercially available ELISA (Serotec, Oxford, UK). The
intra and interassay coefficients of variation were less than 7%. The
assay detection limit was 0.2 ng/ml. No cross-reactivity was observed
with inhibin-A, activin-B, FS or inhibin-B.
Western blot analysis
Proteins were separated on 10% SDS-PAGE under nonreducing
conditions, and blotted on nitrocellulose membrane. Following blocking
with 10% nonfat milk, the membrane was incubated overnight with 0.4
µg/ml of a monoclonal antibody specific for human FS288, 7FS30 (21)
(provided by the NCPIR Technology Development Laboratory) at 4 C. The
membrane was washed in washing buffer (1% nonfat milk in PBS with
0.2% Tween 20) and incubated with a secondary antimouse antibody
coupled to peroxidase (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at a 1/15000 dilution. The membrane
was then washed three times for 30 min in washing buffer, and
immunoreactivity was assessed by chemiluminescence (ECL kit,
Amersham) according to the manufacturer’s instructions.
To address the specificity of the signal, the 7FS30 antibody (0.4
µg/ml) was preincubated for 4 h with purified FS315 (0.8
µg/ml). FS315 was produced by transfection of 293 cells with a cDNA
for human FS315 (kindly provided by Dr. S. Shimasaki) and the
recombinant protein was purified in the NCPIR Technology Development
Laboratory.
Plasmids
Construction of ARE expression vector (pAR6-GFP-Lux). Six
repeats (50 bp each) of the Activin Response Element (ARE) identified
within the Xenopus Mix 2 gene promoter (24) were inserted
upstream from 224 bp of the HSV TK basal promoter (position -211/+12).
To generate the vector pAR6-GFP-Lux, the resulting fragment was
subcloned into pMYC-GFP-LUX-BS (kindly provided by Drs. P. Aftring and
M. W. Freeman), which consisted of a Green Fluorescent Protein
(GFP)-luciferase (Lux) fusion protein with a N-terminal myc tag
subcloned in pBluescript II KS (Stratagene, La Jolla, CA).
This construct was then sequenced to verify the correct positioning of
the ARE and TK elements with the start of the GFP-Lux reporter.
FAST-1 (forkhead activin signal transducer-1) is a DNA-binding protein which mediates activin responsiveness to genes containing the ARE in the Xenopus early embryo (24). The FAST-1 vector consisted of FAST-1 cDNA in pCS2 (kindly provided by Dr. M. Whitman).
Control plasmids
We used pcDNA3-GFP-Lux in which the GFP-luciferase fusion
protein was subcloned into pcDNA3 (Invitrogen, Carlsbad,
CA) as a positive control. TK-GFP-Lux was constructed in the same
manner as the pAR6-GFP-Lux, but without the 6 ARE repeats, as a
negative control. All constructs were confirmed by sequencing.
Transient transfections and luciferase assays
Twenty-four hours before transient transfection experiments,
2 x 105 cells were plated per well in 24-well dishes
and grown in RPMI 1640 supplemented with 10% FBS. Cells were
transfected using lipofectamine (Gibco BRL, Grand Island,
NY) according to the manufacturer’s instructions. In each well, cells
were transfected with the specified constructs in a total amount of 0.4
µg DNA with 1 µl of lipofectamine. Cells were incubated for 5
h with the DNA/lipid complexes in serum-free medium. The medium was
then replaced by RPMI 1640 supplemented with 10% FBS, in the presence
or absence of activin-A. Twenty-four hours after transfection, cells
were lysed with 250 µl of lysis buffer (25 mM
Glycylglycine, pH 7.8, 15 mM MgSO4, 4
mM EGTA, 1 mM dithiothreitol, 1% Triton
X-100). Luminescence was measured using a Monolight 3000 Luminometer
(Analytical Luminescence Laboratory, San Diego, CA) for 10
sec following automated addition of luciferase buffer and 0.625
mM D-luciferin solution (Analytical Luminescence Laboratory, San Diego, CA) to the cell lysates. All
transfection experiments were performed in triplicate and replicated at
least three times. Transfection efficiencies were monitored with
parallel transfections of the pcDNA3-GFP-Lux vector and the luciferase
activities elicited by pcDNA3-GFP-Lux in activin transfected clones and
wild-type cells were normalized to the luciferase activities elicited
by the pcDNA3-GFP-Lux control.
Data analysis
The proliferation of the transfected clones was compared with
that of untransfected PA1 cells by a two-way ANOVA, followed by
Tukey’s multiple comparison test. P < 0.05 was
considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
Notice that free FS levels varied inversely with activin levels and
were undetectable in three clones (C, D, and E).
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). The 32,000 band was
observed in the conditioned media of both cell types after longer
exposure of the blot (data not shown), but in Fig. 1, it can only be
seen in transfected clone E. RhFS 288 exhibited two major bands at
Mr 32,000 and 35,000, whereas rhFS 315 contained two major
bands at Mr 35000 and 38000 (Fig. 1), probably representing
non or monoglycosylated forms of the proteins (13, 25, 26). Antibody
recognition of all FS bands was eliminated by preincubation of 7FS30
with purified FS 315 providing evidence that these bands represent FS
(data not shown). Thus, ßA-transfected PA1 cells
secreting high levels of activin A still secrete FS, most or all of
which is bound to activin, and hence is not detected in the FS SPICA.
Based on the observation that conditioned medium from cultures
containing an equal number of cells gave bands of similar intensity,
the FS secretion appears to be similar in transfected and untransfected
cells (Fig. 1). Moreover, both wild-type and activin transfected PA1
cells secrete multiple forms of FS, most probably representing FS 288,
FS 303, and FS 315 in different glycosylation states.
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In wild-type PA1 cells, radiolabeled activin bound specifically to the
surface, 70% of which was displaceable by heparan sulfate (Fig. 2), indicating activin bound to cell
surface associated FS (14). Simultaneous addition of labeled activin
and a 100-fold excess of unlabeled activin reduced the total binding by
90% (Fig. 2). The difference between heparan sulfate releasable counts
and unlabeled activin displaceable counts represents specific binding
to activin receptors. Thus, at the concentration of labeled activin
used for these experiments, activin binding to cell surface FS exceeds
binding to the activin receptors by 3.5-fold. Addition of 25 ng/ml FS
288 slightly increased the amount of activin binding and, like
endogenous FS, was displaceable by heparan sulfate treatment (Fig. 2).
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). While
this labeled activin was not displaceable by heparan sulfate, it was
displaceable by unlabeled activin (Fig. 2), suggesting that all the
[125I]activin was bound to the receptors while the
cell-surface associated FS was occupied with unlabeled activin secreted
by the ßA-transfected cells. Addition of exogenous FS 288 increased
the amount of labeled activin binding by 5-fold, much greater than the
increase seen in untransfected cells, and these counts were
displaceable by heparan sulfate (Fig. 2), demonstrating the
availability of additional, previously unoccupied, FS binding sites on
ßA transfected PA-1 cells.
Activin secretion by PA1 cells induces a decreased proliferation
rate
Previous reports from our laboratory have shown that exogenous
activin-A decreases the proliferation rate of PA1 cells only after
removal of cell surface FS with heparin pretreatment (21). To
investigate the ability of transfected (endogenous) activin-A to affect
cell proliferation, the proliferation rates of five different activin-A
transfected clones were compared with the proliferation rate of
untransfected PA1 cells. In one representative experiment (Fig. 3A), proliferation, as measured by
3H-thymidine incorporation, was suppressed in activin-A
transfected clones B, C, and E compared with wild-type cells. Figure 3B
shows a composite of four experiments comparing activin’s suppression
of proliferation in five different clones normalized to proliferation
observed in wild-type cells. Clone A, in which activin-A secretion was
low and free FS secretion in the same range as in untransfected cells,
did not show any difference in proliferation compared with
untransfected cells. During the initial characterization of activin
transfected PA-1 clones, additional low secretors similar to clone A
were examined, the results of which were identical to those shown for
clone A (data not shown). Clone B, in which activin secretion was
higher but free FS secretion was still detectable, showed a 32% and
27% decrease in proliferation after 72 h and 96 h of culture
respectively compared with the untransfected cell line, but differences
did not reach statistical significance. Clones C, D, and E, in which
free FS secretion was not detectable, displayed a drastic reduction in
proliferation, up to >70% for clone E. Therefore endogenous
activin affects PA1 cell proliferation without heparin pretreatment.
Furthermore, no difference in proliferation rates were observed when
activin-A transfected clones (clones A and D were tested) were
pretreated with heparan sulfate (data not shown), again indicating that
cell surface FS does not affect the action of endogenous activin.
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). This was also the case for cells
cotransfected with the negative control plasmid TK-GFP-Lux together
with FAST-1 (Fig. 4). When pAR6-GFP-Lux was cotransfected with FAST-1,
substantial basal luciferase activity was observed in PA1 cells, which
could be further induced by exogenous activin-A (Fig. 4). These data
show that PA1 cells contain the required signal transduction machinery
to activate transcription from a Xenopus ARE but are missing
the specific DNA-binding component FAST-1.
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). Maximal
induction of the luciferase activity was achieved at 100 ng/ml
activin-A and was 3-fold higher than in the absence of activin.
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). While
treatment with 100 ng/ml of exogenous activin-A produced a 3-fold
increase in the transcriptional response of wild-type PA1 cells, the
same dose of activin-A had no effect on the response of clone E (Fig. 6). The transcriptional responses of clones C and D were similar to
clone E with a high basal transcriptional activity that was not further
enhanced by treatment with exogenous activin-A (data not shown). Clone
B had a slightly higher basal transcriptional activity than wild-type
cells, which was inducible by 2- to 3-fold by exogenous activin-A,
whereas clone A had a similar transcriptional response to wild-type
cells (data not shown). In one experiment, activin A levels were
measured in the conditioned medium of wild-type PA1 and clone E cells
just before their extraction for the luciferase assay. At that time,
basal levels of activin A were undetectable in PA1 cells and were 11.6
ng/ml in clone E. After treatment with 100 ng/ml of exogenous activin,
activin A levels were similar in wild-type PA1 and clone E cells (68
ng/ml and 71 ng/ml, respectively). Thus, similar levels of
extracellular activin produce different reporter responses, suggesting
that endogenous activin might enhance the effectiveness of the activin
signal transduction system.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine whether the action of endogenous activin might differ from exogenous activin in PA1 cells, we compared the proliferation rate of activin transfected clones to untransfected cells. The present study clearly shows that the overexpression of activin-A in PA1 cells decreases their proliferation. The greatest suppression of proliferation was observed in clones secreting the highest amounts of activin while no effect on proliferation was observed in the low secreting clone (clone A), strongly suggesting that the magnitude of the antiproliferative effect was directly related to the activin expression level. The antiproliferative action of endogenous activin was observed even in the presence of cell-surface FS (i.e. in the absence of heparin treatment).
These results contrast with the absence of an effect in wild-type PA1 cells treated with exogenous activin but without heparin (21, 28). In wild-type PA1 cells at 96 h of culture, the free FS concentration in conditioned medium was higher than in the activin-transfected clones. Similarly, the vast majority of [125I]activin bound to wild-type PA1 cells could be displaced by heparan sulfate, emphasizing that these cells are constitutively coated with FS capable of binding activin in large excess over the binding capacity of their own, cell-surface activin receptors.
On the other hand, in activin transfected clones displaying the lowest proliferation rates and the highest activin secretion (clones C, D, and E), free FS was undetectable in the conditioned medium at 96 h of culture. Moreover, in these clones, the total binding of [125I]activin was much lower than in wild-type PA1 cells but was not displaceable by heparan sulfate. These results are compatible with either a reduction/absence of FS secretion in ßA-transfected PA1 clones, or with cell surface FS being occupied with endogenous activin. In support of the latter possibility, we have previously shown that activin-FS complexes are not dissociable (12) so that unlabeled activin cannot be displaced by [125I]activin once the complexes are formed. In addition, because total FS secretion, as assessed by Western blotting, appeared to be similar in wild-type and in ßA-transfected clones, it is highly likely that in activin-secreting clones, cell surface FS was actually already occupied by endogenous activin.
At this point, cell surface associated FS may no longer be effective in neutralizing the antiproliferative effects of endogenous activin. However, among these clones, the highest activin concentration in the conditioned media at 96 h of culture was 42.4 ± 9.9 ng/ml (clone D). On the other hand, exogenous activin, up to 100 ng/ml was shown to have no effect on the proliferation rate of PA1 cells (28). Therefore it is unlikely that the lack of free FS at the cell surface accounts for all the differences observed between exogenous and endogenous activin on cell proliferation. Taken together with our previous studies on wild-type PA1 cells, these results suggest that endogenous activin (autocrine) can exert its action despite the presence of cell surface associated FS, whereas exogenous activin (paracrine or endocrine) is unable to do so, unless cell surface FS is first removed with heparin.
Using purified rhFS288 and FS315 as relative molecular mass markers, Western blotting experiments demonstrated that PA1 cells secrete multiple forms of FS, most probably 6representative of FS 288, FS 303, and FS 315 in different glycosylation states (13). These results differ slightly from previously published data from our laboratory in which all immunoreactive FS secreted by PA1 cells was shown to be identical to rhFS 288 by Western and ligand blot analysis (21). This can be explained by the fact that in the previous study, FS was isolated from PA1-conditioned medium by sulfate cellufine chromatography, concentrating the C-terminally processed FS forms that display high affinity for heparin-like molecules (14), as opposed to the present study where conditioned medium was examined without purification.
To more directly assess activin signaling in PA1 cells, the transcriptional activation of a reporter gene controlled by an ARE was examined in response to exogenous and endogenous activin. A relatively high basal luciferase activity was observed in wild-type PA1 cells when cotransfected with pAR6-GFP-Lux and FAST-1. Because the basal activity was minimal when the cells were transfected either with the negative control plasmid TK-GFP-Lux together with FAST-1, or with pAR6-GFP-Lux alone, it is unlikely that the high basal activity was due to non specific reporter activation. In two previous studies using the FAST-1/ARE reporter system in mammalian cell lines HepG2 (29) and R1B/liter17 (30), FAST-1 was also required to induce the transcriptional activation of the reporter gene, but in contrast to PA1 cells, the basal reporter activity was very low (29, 30). Because Smad2 and Smad4 are shared by TGFß and activin signaling pathways, the high basal activity observed in PA1 cells when cotransfected with pAR6-GFP-Lux and FAST-1 suggests that these cells secrete some amount of activin or TGFß. As we have been thus far unable to detect natural activin-A protein or ßB mRNAs in these cells, we suspect that endogenous TGFß may be responsible for the high basal activity. Nevertheless, it is clear that transcription of this reporter is increased in response to activin treatment.
Exogenous activin induced a dose responsive transcriptional activation of the pAR6-GFP-Lux in wild-type PA1 cells, whereas it did not affect reporter activities in ßA-transfected cells, suggesting that activin stimulation had already reached a maximum in clones secreting the highest levels of activin. In wild-type PA1 cells, the transcriptional response to exogenous activin was observed even without heparin preincubation. This was in apparent contradiction with the absence of exogenous activin effect on cell proliferation without heparin pretreatment (21). However, we found by [125I]activin binding that lipofectamine, as used in our transient transfection experiments, removed FS from the cell surface in the same manner as heparin (data not shown). Therefore, these experiments cannot address issues pertaining to the role of cell-surface FS in modulating an activin response. However, they show that activin treatment of PA1 cells induces a transcriptional response of the primary activin-signaling pathway under conditions in which proliferation is affected. Therefore, these observations strongly suggest that the proliferation results obtained were due to an activin signaling event.
The same concentration of activin in conditioned medium generated a higher transcriptional response in ßA-transfected cells (endogenous activin) than was observed in untransfected PA1 cells (exogenous activin). One possibility is that activin overexpression in PA1 cells up-regulates one or more activin signaling component(s) which could involve the activin receptor and/or any signaling factor downstream of the receptor in the activin signaling cascade. However, because [125I]activin binding to activin receptors is similar in PA1 untransfected cells and activin secreting clones, it seems unlikely that the higher endogenous activin transcriptional response resulted from up-regulation of the activin receptors. This hyperresponsiveness of the cells to activin in ßA-transfected cells is also consistent with the differences observed between exogenous and endogenous activin in regard to the proliferation rate of PA1 cells: endogenous activin could decrease the proliferation rate of the cells up to 72%, whereas exogenous activin was previously shown to induce a maximum suppression of 30% in the proliferation of wild-type PA-1 cells after heparin pretreatment.
In summary, the present study shows that overexpression of activin in PA1 cells significantly suppresses their proliferation, even in the presence of cell-surface FS. Taken together with our previous studies demonstrating that endogenous cell surface FS, unless first removed by heparin, prevented the antiproliferative effect of exogenous activin in PA1 cells, our results support the concept that FS acts as a barrier for exogenous but not for endogenous activin. Thus cell surface FS could be considered a selective inhibitor for endocrine or paracrine actions, but not for autocrine actions, of activin. It would be of great interest to investigate this model under physiological conditions in which cells express both activin and FS and are also exposed to exogenous activin, such as in granulosa cells.
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Acknowledgments |
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Footnotes |
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Received July 22, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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