Molecular Basis of Bone Morphogenetic Protein-15 Signaling in
Granulosa Cells*
R. Kelly
Moore
,
Fumio
Otsuka§, and
Shunichi
Shimasaki¶
From the Department of Reproductive Medicine, University of
California San Diego, School of Medicine,
La Jolla, California 92093-0633
Received for publication, July 22, 2002, and in revised form, October 8, 2002
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ABSTRACT |
Bone morphogenetic protein-15 (BMP-15), an oocyte
growth factor belonging to the transforming growth factor-
superfamily, has recently been shown to be necessary for normal female
fertility in mammals. We have previously demonstrated that BMP-15
regulates granulosa cell (GC) proliferation and differentiation;
namely, BMP-15 promotes GC mitosis, suppresses follicle-stimulating
hormone (FSH) receptor expression, and stimulates kit ligand
expression. Although the role of BMP-15 in female reproduction has
progressively deserved much attention, there is nothing known to date
about the signaling pathway and receptors for BMP-15. Using rat primary GCs and a human GC cell line, COV434, we have now found that
administration of BMP-15 causes a rapid and transient phosphorylation,
thus activation, of the Smad1/5/8 pathway. BMP-15 also stimulated
promoter activity of a selective BMP-responsive reporter construct,
further demonstrating the stimulation of Smad1/5/8 signaling by BMP-15.
In contrast, BMP-15 stimulation of Smad2 phosphorylation was very weak.
To identify the receptors for BMP-15, we utilized recombinant
extracellular domains of individual transforming growth factor-
superfamily receptors and found that activin receptor-like kinase-6
extracellular domain most effectively co-immunoprecipitates with
BMP-15, whereas BMP receptor type II extracellular domain was most
effective in inhibiting BMP-15 bioactivity on FSH-induced progesterone
production and GC thymidine incorporation. We also investigated whether
activation of the MAPK pathway is necessary for BMP-15 biological
activity and found that the addition of U0126, an inhibitor of ERK1/2
phosphorylation, suppresses BMP-15 activity on GC mitotsis but not on
FSH-induced progesterone production, suggesting a selective signaling
cascade in GC proliferation and differentiation.
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INTRODUCTION |
Bone morphogenetic protein-15
(BMP-15),1 also called growth
and differentiation factor-9B (GDF-9B), is a recently discovered oocyte-derived growth factor and a member of the transforming growth
factor-
(TGF-
) superfamily (1, 2). BMP-15 has been shown to play
integral roles in the regulation of ovarian folliculogenesis and
follicular function (3). Our previous in vitro studies in
rats using recombinant BMP-15 identified granulosa cells (GCs) as the
predominant target cell type for BMP-15 (4). These studies also
identified three major biological functions of BMP-15 in the ovary
(4-6). (i) BMP-15 is a potent stimulator of GC mitosis; (ii) BMP-15
inhibits the expression of FSH receptor mRNA in GCs, resulting in
the subsequent suppression of FSH-induced progesterone synthesis as
well as FSH-induced expression of a battery of mRNAs in GCs; and
(iii) BMP-15 stimulates the expression of kit ligand mRNA in GCs.
Furthermore, the biological activity of BMP-15 was found to be
inhibited by follistatin, which is highly expressed by GCs in
developing follicles (7, 8). The importance of BMP-15 in fertility in
sheep was exemplified by the demonstration that naturally occurring
mutations in the bmp15 gene in Inverdale (FecXI)
and Hanna (FecXH) ewes caused an increase in the ovulation
rate in heterozygotes due, in part, to an increased FSH sensitivity of
GCs but caused infertility in homozygotes due to a block in the primary
stage of folliculogenesis (9). Mice with targeted deletions in the bmp15 gene are subfertile, demonstrating the importance of
BMP-15 in fertility in the mouse as well (10).
The TGF-
superfamily consists of over 30 members, which are
classified into the superfamily based upon structural homology (1, 11).
A common feature of most of the members of the TGF-
superfamily is
the presence of seven conserved cysteine residues, six of which form a
characteristic "cysteine knot" structure and one of which forms a
disulfide bridge between two subunits, making the molecule a covalently
linked dimer (12). Interestingly, BMP-15 and its closest homologue,
GDF-9, which is also an oocyte-derived factor, are unique among the
TGF-
superfamily in that they lack the conserved fourth cysteine,
which is responsible for the dimerization of the subunits (1). Thus, it
is not known whether BMP-15 exists as noncovalently linked dimers or as monomers.
Despite the established importance of BMP-15 in female fertility, the
receptors and signaling pathways for BMP-15 have yet to be identified.
Receptors for the majority of TGF-
superfamily members consist of
two closely related subsets of transmembrane serine/threonine kinase
receptors, the type I and type II receptors (13). To date, seven type I
and five type II receptors have been identified in mammals. Upon
binding of dimeric TGF-
superfamily members to specific type I and
type II receptors, the receptor complexes cause the phosphorylation of
intracellular signaling molecules called Smads, which then translocate
to the nucleus and regulate transcription of target genes (14-16). The
receptor-regulated Smads can be grouped into two subsets; Smad2 and
Smad3 are activated by TGF-
and activin, whereas Smad1, Smad5, and
Smad8 are activated by BMPs. For this signaling to occur, the ligand
must bind both type I and type II receptors, but the specificity of the
Smad signaling pathway, which is stimulated by a particular TGF-
superfamily ligand, is determined by the type I receptor (17). In
addition to Smad signaling, recent studies have demonstrated that the
mitogen-activated protein kinase (MAPK) family of signaling molecules
can modulate the signal transduction of TGF-
superfamily members
through cross-talk with the Smad pathway in certain physiological
circumstances (18).
Currently, there is nothing known about the receptors or signaling
pathway of BMP-15. Given BMP-15's unique structure compared with other
TGF-
superfamily members, it is possible that BMP-15 uses unique
receptors and signaling; however, it has been speculated that BMP-15
may use one or more of the established activin, TGF-
, or BMP
receptors and may stimulate a subsequent Smad signaling pathway
(19-21). The presence of activin receptors, which include ActR-II,
ActR-IIB, and activin receptor-like kinase-4 (ALK-4, also called
ActR-IB) and BMP receptors, which include ActR-II, BMPR-II, ALK-3
(BMPR-IA), ALK-6 (BMPR-IB), and ALK-2 (ActR-IA), as well as Smad
molecules have been identified in GCs in various mammalian species (19,
20, 22-29); however, there is a paucity of reports that demonstrate
the functional activation of these pathways in any GC model. Of the
MAPK molecules, extracellular signal-regulated kinase 1/2 (ERK1/2) are
expressed in GCs and have been most closely associated with the
regulation of GC function (30-34).
In the present study, we investigate whether BMP-15 can use previously
identified TGF-
superfamily receptors and can activate specific Smad
signaling pathways. We also investigate whether ERK1/2 signaling
molecules play a role in mediating the biological activities of BMP-15.
For these studies, we use two GC models, rat primary GCs from early
antral follicles and a human GC cell line, COV434 (35). The rat primary
GC model has been well established and used to elucidate the biological
activity of recombinant BMP-15 by our laboratory (4-6). COV434 cells
are an immortalized human GC cell line that has retained the ability to
produce estradiol upon FSH stimulation, thus demonstrating the presence
of functional FSH receptors (36).
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EXPERIMENTAL PROCEDURES |
Reagents and Supplies--
Diethystilbestrol was purchased from
Sigma, and U0126, an inhibitor of ERK1/2 phosphorylation, was from
Promega Corp. (Madison, WI). Female Sprague-Dawley rats were from
Charles River Laboratories (Wilmington, MA). Recombinant extracellular
domains (ECDs) fused to the Fc region of human IgG (receptor-ECD/Fc
chimera) of human BMPR-II, ActR-II, ALK-3 (BMPR-IA), ALK-6 (BMPR-IB),
and ALK-2 (ActR-IA) were purchased from R & D Systems (Minneapolis,
MN). Recombinant human BMP-6 and BMP-7 were generously provided by Dr.
Kuber Sampath. Recombinant rat activin-A was produced by Chinese hamster ovary cells and purified using a follistatin affinity column
followed by two steps of reverse-phase high pressure liquid chromatography (6). Recombinant human BMP-15 tagged with a FLAG epitope
was produced by 293 cells and purified using anti-FLAG M2 Affinity Gel
(Sigma) as reported previously (4).
Primary Cell Culture--
Female Sprague-Dawley rats (23 days
old) were implanted with silastic capsules containing 10 mg of
diethystilbestrol to increase GC number. After 4 days of
diethystilbestrol exposure, GCs were collected from the ovaries and
cultured in serum-free McCoy's 5a medium supplemented with 2 mM L-glutamine and antibiotics at 37 °C in
an atmosphere of 5% CO2 in air. All animal protocols were
approved by the University of California at San Diego Institutional Animal Care and Use Committee.
Immunoblot Analysis of Phospho-Smad Proteins--
GCs (1 × 106 viable cells) were seeded in a 24-well plate. After a
5-h preculture, BMP-6, -7, or -15 or activin-A was added to the culture
medium. COV434 cells (generously provided by Dr. Peter I. Schrier) were
seeded in a 24-well plate and were maintained in high glucose
Dulbecco's modified Eagle's medium containing 10% fetal bovine
serum. Once the cells became 80% confluent, the medium was replaced
with serum-free Dulbecco's modified Eagle's medium. After a 3-h
preculture in serum-free medium, BMP-6, -7, or -15 or activin-A was
added to the culture medium. Following culture for the indicated
periods of time, both GCs and COV434 cells were solubilized in 100 µl
of radioimmune precipitation lysis buffer (Upstate Biotechnology, Inc.,
Lake Placid, NY) containing Protease-Arrest (Geno Technology, Inc., St.
Louis, MO), 1 mM Na3VO4, 1 mM NaF, 2% SDS, and 4%
-mercaptoethanol. Lysates were
then subjected to SDS-PAGE/immunoblotting analysis using two previously validated rabbit antisera, anti-phospho-Smad1 (PS-1) and
anti-phospho-Smad2 (PS-2), that were generously provided by Dr.
Carl-Henrik Heldin. PS-1 cross-reacts specifically with phospho-Smad1,
-5, and -8 (the BMP-responsive Smads), and PS-2, which is specific for
phospho-Smad2, does not cross-react with phospho-Smad3 (37). In order
to confirm that there were no differences in the levels of total
protein between groups, antisera that detect total ERK1/2 were used for SDS-PAGE/immunoblotting analysis as described previously (30) (data not shown).
Transient Transfection and Luciferase Assay--
COV434 cells
were seeded in a 24-well dish and cultured in medium containing
10% fetal bovine serum. When the cells became 60-70% confluent, they
were co-transfected with 500 ng of pXVent2-Luc (kindly provided by Dr.
Joan Massagué) and 50 ng of cytomegalovirus-
-galactosidase plasmid (pCMV-
-gal) using FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Basel, Switzerland). 12 h after
transfection, the culture media were replaced with 1% fetal bovine
serum medium, and BMP-15 was added to a final concentration of 100 ng/ml. After culture for 24 h, the cells were washed with
phosphate-buffered saline (PBS) and then lysed with 60 µl of cell
culture lysis reagent (Promega Corp., Madison, WI). Luciferase activity
in 20 µl of the cell lysate was measured with a TD-20/20 Luminometer
(Turner Designs, Sunnyvale, CA) using Luciferase Assay Reagent (Promega Corp., Madison, WI).
-Galactosidase activity in 10 µl of the lysate was measured using the Galacto-Light Plus System (Applied Biosystems, Bedford, MA) and used to normalize transfection
efficiencies. The data are shown as the ratio of luciferase to
-galactosidase activity and expressed as the -fold change as
compared with non-BMP-15-treated (control) cells.
Co-immunoprecipitation of BMP-15 and Receptor ECD/Fc
Chimera--
BMP-15 (100 ng/ml) was mixed with the indicated
receptor-ECD/Fc chimera proteins (2 µg/ml) in PBS to a total volume
of 250 µl and incubated at 4 °C for 4 h. Anti-FLAG monoclonal
antibodies conjugated to agarose beads (anti-FLAG M2 affinity gel; 10 µl) in 300 µl of PBS were added, and the incubation was continued for another 2 h at 4 °C. Following incubation, the agarose
beads were centrifuged and washed five times with 1 ml of PBS. Proteins bound to the agarose beads were eluted with SDS-PAGE sample buffer and
subjected to SDS-PAGE fractionation and immunoblotting analysis using a
horseradish peroxidase-conjugated goat anti-human IgG antiserum to
detect the receptor/Fc chimera proteins.
Analysis of Steroid Production--
GCs (105 viable
cells) were cultured in a 96-well plate with 200 µl of serum-free
medium containing one or a combination of FSH, BMP-15, various
receptor-ECD/Fc chimeras, and U0126. After a 48-h culture, the
culture media were collected and stored at
20 °C until assay. The
levels of progesterone in the medium were measured by standard
radioimmunoassay procedures (4).
Thymidine Incorporation Assay--
GCs (2 × 105 viable cells) were cultured in 1.5-ml polypropylene
tubes containing 200 µl (final volume) of serum-free culture medium.
After a 24-h preculture period, cells were incubated with 0.5 µCi/tube of [methyl-3H]thymidine either
alone or with a combination of BMP-15 and individual TGF-
superfamily receptor/Fc chimera or U0126. After another 24 h of
culture, the GCs were washed with PBS, centrifuged (2000 × g, 30 min), and incubated with 10% ice-cold trichloroacetic acid for 30 min at 4 °C. The cell pellet was solubilized in 0.2 M NaOH, and its radioactivity was measured (4).
Statistical Analysis--
All results shown are mean ± S.E. of at least three separate experiments with triplicate
determinations for each treatment. Differences between groups were
analyzed for statistical significance using analysis of variance (SPSS
Standard version 10.0.1, SPSS Inc., Chicago, IL). p values
of <0.05 were accepted as statistically significant.
 |
RESULTS |
We have previously demonstrated that recombinant BMP-15 exhibits
specific biological activities in rat primary GCs (4-6). To
investigate the signaling pathways of BMP-15, we first subjected lysates of rat primary GCs that were exposed to BMP-15, BMP-6, BMP-7,
or activin-A to SDS-PAGE/immunoblotting analysis using PS-1 antiserum,
which has been validated to specifically detect phospho-Smad1/5/8.
Following a 1-h exposure of GCs to BMP-15 (100 ng/ml), there was a
pronounced increase in phospho-Smad1/5/8, which was also observed in
the lysates of GCs treated with 100 ng/ml BMP-6 and BMP-7 (Fig.
1A). In contrast, lysates from
untreated (control) GCs and GCs treated with 100 ng/ml activin-A did
not display any significant amount of phospho-Smad1/5/8. A similar stimulation of the Smad1/5/8 pathway by BMP-15, BMP-6, and BMP-7, but
not by activin-A, was observed in a human GC cell line, COV434 (Fig.
1B). The same phospho-Smad1/5/8 response to these growth factors was also observed in CHO-K1 cells (data not shown), indicating that BMP-15 stimulation of this pathway is not restricted to GCs. To
further characterize the BMP-15 response in COV434 cells, we analyzed
the time course of the presence of phospho-Smad1/5/8 following BMP-15
administration. The presence of phospho-Smad1/5/8 was evident as early
as 15 min after BMP-15 administration, increased at 30 min, and peaked
at 1 h, and by 4 h it was barely detectable (Fig.
2).

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Fig. 1.
Activation of the Smad1/5/8 signaling pathway
in rat primary GCs and COV434 cells. Ligands were added to the
medium for the culture of rat primary GCs (A) and
COV434 cells (B). After culture for 1 h, cells were
lysed and subjected to SDS-PAGE/immunoblotting using PS-1
antiserum. Numbers in parentheses
represent the concentration of ligands in ng/ml.
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Fig. 2.
Time course of PS-1 activation by BMP-15 in
COV434 cells. Following treatment with BMP-15 (100 ng/ml) for the
indicated incubation periods, cells were lysed and subjected to
SDS-PAGE/immunoblotting using PS-1 antiserum.
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The lysates from primary rat GCs and COV434 cells exposed to BMP-15,
BMP-6, BMP-7, or activin-A were also subjected to
SDS-PAGE/immunoblotting using the PS-2 antiserum, which is specific for
phosphorylated Smad2. In the lysates of rat primary GCs treated with
activin-A, a very weak PS-2 signal was detectable, whereas the PS-2
signals in cells treated with BMP-15, BMP-6, and BMP-7 were
indistinguishable from that of untreated controls (data not shown).
However, in the lysates from COV434 cells that were exposed to
activin-A, there was a pronounced increase in phospho-Smad2, whereas
treatment with BMP-15, -6, and -7 had little effect on levels of
phospho-Smad2 (Fig. 3).

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Fig. 3.
Activation of the Smad2 signaling pathway in
COV434 cells. Ligands were added to COV434 culture medium.
After culture for 1 h, cells were lysed and subjected to
SDS-PAGE/immunoblotting with PS-2 antiserum. Numbers
in parentheses represent the concentration of ligands
in ng/ml.
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To further demonstrate the functional activation of the Smad1/5/8
pathway by BMP-15, COV434 cells were transiently transfected with a
transcriptional reporter construct, pXVent2-Luc, which has been shown
to specifically respond to BMP signaling in P19 cells, C3H10T1/2 cells,
and Xenopus embryos (38-40). Treatment with BMP-15 at 100 ng/ml caused a 3.3-fold increase in XVent2-luciferase activity as
compared with untreated controls (Fig.
4).

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Fig. 4.
Activation of XVent2 luciferase activity by
BMP-15 in COV434 cells. COV434 cells were transiently transfected
with the BMP-responsive reporter construct, pXVent2-Luc. After 24-h
incubation with or without BMP-15, cells were lysed, and luciferase was
measured. An asterisk denotes a significant difference at
p < 0.05.
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Of the TGF-
superfamily receptors identified to date, BMPR-II,
ActR-II, ALK-3, ALK-6, and ALK-2 have been implicated in the activation
of the Smad1/5/8 pathway (14); thus, we hypothesized that one or more
of these receptors may be involved in BMP-15 activation of the
Smad1/5/8 pathway. To characterize biochemical binding ability, we used
receptor-ECD/Fc chimera proteins for co-immunoprecipitation studies
with BMP-15. Using a monoclonal anti-FLAG antibody conjugated to
agarose beads, we analyzed the ability of BMP-15 to bind each
receptor-ECD. SDS-PAGE/immunoblot analysis of the receptor-ECD chimera
before immunoprecipitation showed that there was a similar amount of
immunoreactive ECD in each group and that the individual receptor-ECDs
had unique SDS-PAGE migration patterns (Fig.
5A). The BMPR-II-ECD migrates
at 65-75 kDa, ActR-II-ECD migrates at 50-60 kDa, ALK-3-ECD migrates
at 55-65 kDa, and ALK-6-ECD and ALK-2-ECD migrate at 50-55 kDa.
Co-immunoprecipitation studies with BMP-15 revealed that, of the five
candidate receptor-ECD chimeras, ALK-6 co-immunoprecipitation was
clearly higher than the other candidates (Fig. 5B). The
relative ability of each receptor-ECD to co-immunoprecipitate with
BMP-15 was found to be approximately as follows: ALK-6-ECD
ALK-2-ECD = ActR-II-ECD > BMPR-II-ECD
ALK-3-ECD. In
order to ensure that the co-immunoprecipitation was due to the binding
of the receptor-ECDs to BMP-15, and not directly to the
anti-FLAG-agarose, the receptor-ECDs were immunoprecipitated in the
absence of BMP-15. In these studies, there was no detectable immunoprecipitated receptor-ECDs, indicating that the observed co-immunoprecipitation was due to the specific binding of the receptor-ECDs to BMP-15.

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Fig. 5.
Co-immunoprecipitation of specific receptor
ECD/Fc chimera and BMP-15. A, the same amount of the
indicated receptor-ECD was subjected to SDS-PAGE/immunoblotting.
B, the indicated receptor-ECDs were co-immunoprecipitated
with BMP-15 using an anti-FLAG monoclonal antibody conjugated to
agarose beads. Following elution from the agarose beads, receptor ECDs
were subjected to SDS-PAGE/immunoblotting as in the upper
panel.
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We next used the receptor-ECD/Fc chimera to determine whether these
proteins can inhibit the biological effects of BMP-15 in rat primary
GCs. To this end, we first evaluated the effects of BMP-15 on
FSH-induced progesterone production in the presence and absence of the
receptor-ECD/Fc chimera (Fig. 6). The
addition of BMP-15 (30 ng/ml) caused a 38% drop in FSH-induced
progesterone production compared with FSH alone. Interestingly, the
inhibitory effect of BMP-15 on FSH-induced progesterone synthesis was
almost completely abolished by BMPR-II-ECD. ActR-II-ECD and ALK-6-ECD also reversed the effects of BMP-15; however, their effects were not as
pronounced as BMPR-II-ECD. ALK-3-ECD had no effect on BMP-15 suppression of FSH-induced progesterone production. In addition, although not shown, ALK-2-ECD alone (in the absence of BMP-15) suppressed FSH-induced progesterone synthesis by 46% and had no observable effect on BMP-15 action; therefore, it was not possible to
determine the effects of this receptor-ECD on BMP-15 steroidogenic properties.

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Fig. 6.
Effect of receptor-ECDs on BMP-15 suppression
of FSH-induced progesterone production. Rat primary GCs were
cultured with FSH (3 ng/ml) in the absence or presence of BMP-15.
Specific receptor-ECDs (1 µg/ml) were added to the culture
medium with BMP-15 as indicated. Following a 48-h culture,
progesterone levels in the culture medium were measured by a
specific radioimmunoassay. * and **, group means are significantly
different from FSH alone levels at p < 0.05 and at
p < 0.001, respectively.
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To further evaluate the specific ability of the receptor-ECDs to
inhibit BMP-15 biological activity, we performed a thymidine incorporation assay. In this analysis, similar to the steroid assay,
BMPR-II-ECD was most effective in inhibiting BMP-15 biological activity
(Fig. 7). To verify the specificity of
the receptor-ECDs in this system, we examined the ability of these
receptor-ECDs to inhibit thymidine incorporation induced by BMP-7 and
activin-A. BMPR-II-ECD was most effective in inhibiting BMP-7-induced
thymidine incorporation followed by ActR-II-ECD. The type I receptor
ECDs were less effective than the type II receptor-ECDs. In contrast, ActR-II-ECD completely inhibited activin-A-induced thymidine
incorporation, whereas the other receptor-ECDs had only a small effect.
Since the type II receptor-ECDs were more effective than the type I receptor-ECDs in inhibiting thymidine incorporation induced by BMP-15,
BMP-7, and activin-A, we compared the dose-response effects of these
receptor-ECDs using the thymidine incorporation assays (Fig.
8). BMPR-II-ECD inhibited BMP-15
biological activity in a dose-dependent manner and was
significantly more potent than ActR-II-ECD; BMP-7-induced thymidine
incorporation was equally inhibited by BMPR-II-ECD and ActR-II-ECD;
and ActR-II-ECD was significantly more effective in inhibiting
activin-A biological activity than BMPR-II-ECD.

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Fig. 7.
Effect of receptor-ECDs on thymidine
incorporation induced by BMP-15, BMP-7, and activin-A. Rat primary
GCs were cultured with [methyl-3H]thymidine
either in the absence (control) or presence of BMP-15 (A),
BMP-7 (B), or activin-A (C) with or without
specific receptor-ECDs (1 µg/ml). Following a 24-h culture, the
labeled thymidine incorporated into the cells was counted.
Bars with different letters indicate
that group means are significantly different from each other at
p < 0.05.
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Fig. 8.
Dose-dependent effects of type II
receptor-ECDs on thymidine incorporation induced by BMP-15, BMP-7, and
activin-A. Rat primary GCs were cultured with
[methyl-3H]thymidine in the absence or
presence of activin-A (100 ng/ml), BMP-7 (500 ng/ml), or BMP-15 (100 ng/ml) with either BMPR-II-ECD (shaded circles)
or ActR-II-ECD (open circles) at the indicated
concentrations. Following a 24-h culture, the labeled thymidine
incorporated into the cells was counted. The asterisks
indicate significant differences (p < 0.05) in
thymidine incorporation between BMPR-II-ECD-treated and
ActR-II-ECD-treated cells.
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Since ERK1/2 signaling has been shown to act either as a positive
transducer of BMP signaling or as an antagonist of BMP-induced Smad
signaling, depending on the cell type (18), we investigated whether
ERK1/2 molecules also play a role in BMP-15 signaling in GCs using
U0126, a selective inhibitor of ERK1/2 phosphorylation. The addition of
U0126 (3 µM) to rat primary GC cultures had no significant effect on basal levels of thymidine incorporation but did
cause a reduction of BMP-15-induced thymidine incorporation when added
at 1 and 3 µM (Fig. 9). We
further determined whether inhibition of ERK1/2 phosphorylation also
mediates the effects of BMP-15 on GC progesterone production. As
previously reported (30), the addition of U0126 (3 µM)
inhibited FSH-induced progesterone production, as did BMP-15 (100 ng/ml) (Fig. 10). However, U0126 had no
effect on BMP-15 inhibition of progesterone synthesis.

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Fig. 9.
Effect of U0126 on thymidine incorporation
induced by BMP-15. Rat primary GCs were cultured with
[methyl-3H]thymidine either in the absence
(control) or presence of BMP-15 with or without U0126 at the indicated
concentrations. Following a 24-h culture, the labeled thymidine
incorporated into the cells was counted. Bars with
different letters indicate that group means are
significantly different from each other at p < 0.05.
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Fig. 10.
Effect of U0126 on BMP-15 suppression of
FSH-induced progesterone production. Rat primary GCs were cultured
with FSH (10 ng/ml) in the absence or presence of BMP-15 (100 ng/ml)
and U0126 (3 µM). Following a 48-h culture, progesterone
levels in the culture medium were measured by a specific
radioimmunoassay. Bars with different
letters indicate that group means are significantly
different from each other at p < 0.05.
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 |
DISCUSSION |
The majority of TGF-
superfamily members use intracellular
second messenger molecules called Smads as a major component of their
signaling pathway. In this pathway, the TGF-
superfamily ligands
first bind to specific serine/threonine kinase receptors, which then
phosphorylate (activate) ubiquitously expressed
"pathway-restricted" Smad molecules. These pathway-restricted Smads
can be classified into two groups based on the subfamily of ligands
that can activate specific Smad molecules. Smad2 and Smad3 are
considered activin/TGF-
-responsive Smads, because the addition of
the activins and the TGF-
s cause the activation of these Smads.
Nodal, another TGF-
superfamily member, has also been shown to use
Smad2/3 for its signaling (41). Smad1, Smad5, and Smad8 are considered
to be BMP-responsive Smads, because most BMPs identified to date,
including BMP-2, -4, -6, and -7, have been shown to activate this Smad
pathway in a number of different cell types (17, 42, 43). Other ligands
that use the Smad1/5/8 pathway include GDF-5 (44) and Müllerian inhibiting substance (40, 45, 46). Based on the fact that most TGF-
superfamily members can activate one of these signaling pathways, we investigated whether BMP-15 activates the Smad1/5/8 or the
Smad2/3 pathway. Treatment of both rat primary GCs and a human GC cell
line, COV 434, with BMP-6, BMP-7, and also BMP-15 caused a clear
increase in the levels of phosphorylated (activated) Smad1/5/8 as
determined by immunoblotting using the PS-1 antiserum. These data
represent the first demonstration of BMP-15 signaling in any cell type.
Although the activation of the Smad1/5/8 pathway by BMP-6 and BMP-7 has
been well documented in a number of cell types (17, 42, 43), this is
also the first demonstration of the activation of this pathway by any
ligand in a GC model. The rapid and transient time course of BMP-15
action on levels of phospho-Smads was consistent with a direct effect
of BMP-15 and was similar to that reported for other BMPs in various
cell types. To verify the functional activation of the Smad1/5/8
pathway by BMP-15, we transiently transfected COV434 cells with the
BMP-responsive reporter, pXVent2-Luc. The administration of BMP-15
caused a significant increase in the luciferase activity by this
reporter, indicating that the Smad proteins activated by BMP-15 can
transduce their signals to activate transcription of the target gene.
Of the various TGF-
superfamily members, ActR-II, BMPR-II, ALK-3,
ALK-6, and ALK-2 have been shown to be directly involved in the
stimulation of the Smad1/5/8 signaling pathway in many cell types (14).
Given that BMP-15 activates this pathway in GCs and that each of these
receptors is expressed in GCs, we hypothesized that it is likely that
BMP-15 may have an affinity for one or more of these receptors. To
identify biochemical binding ability, we utilized the ECD/Fc chimera of
these receptors and attempted to co-immunoprecipitate with BMP-15. Of
these ECDs, ALK-6-ECD was most efficiently co-immunoprecipitated with
BMP-15. In contrast, ALK-3-ECD exhibited a very low ability to
co-immunoprecipitate with BMP-15. BMPR-II-ECD, ActR-II-ECD, and
ALK-2-ECD all clearly co-immunoprecipitated with BMP-15, but the degree
of co-immunoprecipitation was substantially less than that of
ALK-6-ECD. BMPs can be grouped into subcategories based on their
relative affinities for specific type I receptors. In this model, it
has been shown that BMP-4 and GDF-5 preferentially bind ALK-3 and/or
ALK-6, whereas BMP-6 and -7 preferentially bind ALK-2 and/or ALK-6 (17,
43, 47). Because of the low ability of BMP-15 to bind ALK-3, BMP-15
signaling probably more closely resembles BMP-6 and -7 than that of
BMP-4 and GDF-5.
In contrast to the relatively low biochemical binding ability of the
BMPR-II-ECD to BMP-15 identified by co-immunoprecipitation, the
BMPR-II-ECD was most effective in inhibiting the effects of BMP-15 on
both FSH-induced progesterone production and thymidine incorporation. A
characteristic feature of the interactions of TGF-
superfamily
ligands and their component receptors is that the choice of receptors
for a given ligand depends on the presence and amount of type I and
type II receptors available. As a result, the particular receptor
subset that a ligand preferentially binds can differ, depending on the
cell type (43). In this regard, a number of studies have shown that,
when singly overexpressed on the cell surface, BMPR-II has very low or
no affinity for BMP-2, -4, -6, and -7, whereas when co-overexpressed
with the appropriate type I receptors, the affinity of these BMPs for
BMPR-II increases dramatically. In contrast, BMPs do bind to type I
receptors when singly overexpressed (48-51). According to this nature
of the interactions between BMP ligands and their receptors, it would
be expected that BMPs may first bind to the type I receptors followed
by the recruitment of type II receptors (50, 51). Therefore, it seems likely that in the GC cultures, BMP-15 first binds to the highly expressed endogenous ALK-6 receptors on the surface of the GCs (23),
which causes a conformational change in the ALK-6 receptor structure,
which then facilitates the BMP-15·ALK-6 complex to recruit
BMPR-II, consequently eliciting BMP-15 biological activity (Fig.
11A). Consistent with this
model, our data in this study could be explained by the mechanism that
BMP-15 preferentially binds the highly expressed intact ALK-6 on the
surface of GCs, as compared with the exogenously added ALK-6-ECD (Fig.
11B). Also in accordance with this model, the ability of
BMPR-II-ECD to inhibit BMP-15 biological activity could have been
elicited by its ability to bind to the BMP-15·ALK-6 complex and
disrupt the recruitment of the endogenous BMPR-II and subsequent
signaling (Fig. 11C).

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|
Fig. 11.
Proposed mechanism of the interaction
between BMP-15 and its receptors. A, in the absence of
receptor-ECDs, BMP-15 binds to the highly expressed ALK-6 receptors on
the surface of the GCs. After a conformational change in the ALK-6
receptor, the BMP-15·ALK-6 complex recruits BMPR-II, resulting in
signal transduction (Smad1/5/8 phosphorylation). B, BMP-15
binds ALK-6-ECD in a GC-free system; however, in the presence of GCs,
BMP-15 predominantly binds to cell surface ALK-6 receptors.
C, BMPR-II-ECD effectively binds to the BMP-15·ALK-6
complex formed on the GC surface, which inhibits the recruitment of the
endogenous BMPR-II receptors, thus inhibiting subsequent BMP-15
signaling and biological activity.
|
|
Although the Smad pathway is the most studied signaling pathway for BMP
ligands, there are many recent reports identifying alternative
signaling pathways that mediate biological actions of BMPs in various
cell types (52). In particular, the MAPK family of signaling molecules,
including ERK1/2, p38, and stress-activated protein kinases/Jun
N-terminal kinases, have been shown to exhibit cross-talk with the Smad
pathway in the intracellular transduction of BMP signals.
Interestingly, activated MAPK molecules have been shown to either
positively transduce BMP/Smad signals or to act as an inhibitor of Smad
signaling. Of these MAPK molecules, ERK1/2 have recently been
implicated in the regulation of GC function (30-33). To further
characterize the signaling of BMP-15 in rat primary GCs, we evaluated
whether a specific inhibitor of ERK1/2 phosphorylation, U0126, has any
effect on BMP-15-induced thymidine incorporation and BMP-15 inhibition
of progesterone synthesis. The inhibition of BMP-15-induced GC
thymidine incorporation by U0126 demonstrates that ERK1/2 signaling is
in fact necessary for this biological activity of BMP-15. A recent
report by the Eppig laboratory (34) demonstrated that U0126 also
inhibits cumulus GC expansion induced by GDF-9, the growth factor that most closely resembles BMP-15, suggesting that both growth factors may
share the ERK1/2 signaling pathway in GCs to exert certain biological
activities. In contrast to the inhibition of BMP-15 action on GC
mitosis, U0126 had no effect on BMP-15 suppression of FSH-induced
progesterone production, demonstrating that activation of the ERK1/2
pathway is not necessary for this action of BMP-15. These findings
indicate that there is a divergence in the signal transduction pathways
regulating steriodogenesis and mitosis by BMP-15.
In summary, the present studies identify that BMP-15 activates the
Smad1/5/8 pathway in two GC models, rat primary GCs from early antral
follicles and a human GC cell line, COV434. We also demonstrate that
BMP-15 co-immunoprecipitates with ALK-6-ECD more efficiently than other
BMP receptor-ECDs and that BMPR-II-ECD is most efficient in inhibiting
BMP-15 biological activities in rat primary GCs. Additionally, we show
that activated ERK1/2 molecules are necessary for BMP-15 action on GC
mitosis but not for BMP-15 action on FSH-induced progesterone
synthesis, suggesting that there are selective pathways for BMP-15
regulation of GC proliferation and differentiation.
 |
ACKNOWLEDGEMENTS |
We thank Mei Wang for excellent technical
assistance. We also thank Dr. Kuber Sampath for recombinant BMP-6 and
BMP-7, Dr. Peter I. Schrier for COV434 cells, Dr. Carl-Henrik Heldin
for PS-1 and PS-2 antisera, and Dr. Joan Massagué for
pXVent2-Luc.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health (NIH) Grant RO1 HD41494 and NICHD, NIH, cooperative agreement U54HD12303 as part of the Specialized Cooperative Centers Program in
Reproduction Research and the University of California at San Diego
Academic Senate (Grant RA 882M).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Supported by NIH Grant F32 HD41320.
§
Supported by a fellowship grant from the Lalor Foundation.
¶
To whom correspondence should be addressed: Dept. of
Reproductive Medicine, University of California at San Diego,
School of Medicine, 9500 Gilman Dr., La Jolla, CA 92093-0633. E-mail: sshimasaki@ucsd.edu.
Published, JBC Papers in Press, November 4, 2002, DOI 10.1074/jbc.M207362200
 |
ABBREVIATIONS |
The abbreviations used are:
BMP, bone
morphogenetic protein;
ALK, activin receptor-like kinase;
ECD, extracellular domain;
ERK1/2, extracellular signal-regulated kinase
1/2;
FSH, follicle-stimulating hormone;
GC, granulosa cell;
GDF, growth and differentiation factor;
MAPK, mitogen-activated protein
kinase;
TGF-
, transforming growth factor-
;
PBS, phosphate-buffered saline.
 |
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