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INTRODUCTION |
Activins, inhibins, and
BMPs1 are structurally
related members of the TGF
superfamily. BMPs are a large family with
extensive and extremely complex roles both in development and adult
life (1-3). For example, BMP-7, 1 of more than 20 BMPs, has roles in
kidney morphogenesis and bone formation during development (4, 5) and
is involved in regulating gonadal function in the adult (6, 7). BMPs
stimulate target cells by assembling a cell surface complex containing
type II and type I receptors (8). In this receptor complex, type II
receptors activate type I receptors by phosphorylating the GS domain of
the type I receptor. The activated type I receptors then activate Smad
proteins, such as Smad1, which transduce signals into the cell nucleus
(9, 10). Although BMP type I receptors (including ALK-2, ALK-3, and
ALK-6) are largely specific for the BMP family, this is not true for
type II receptors. BMPRII binds only BMPs (11-13), but ActRII and
ActRIIB can bind both BMPs and activins (14) and can mediate signaling
by either family. Individual BMPs may bind preferentially to specific
type I or type II receptors, as illustrated by the preferentially
binding of BMP-7 to ActRII (14) and ALK-2 (15, 16). In general,
however, the BMP family as a whole makes use of all of these type I and
type II receptors.
Activins and inhibins were first identified as regulators of
reproduction that antagonistically modulate the endocrine interaction of the pituitary and gonadal systems. Activins are local regulators of
pituitary FSH release, whereas inhibins are produced by the gonads in
response to FSH and act at the pituitary to attenuate activin effects
(17). Activins, like BMPs, stimulate target cells by assembling
receptor complexes containing type I and type II receptors at the cell
membrane. In these ligand-receptor complexes, distinct activin-specific
type I receptors are activated and in turn activate activin-specific
Smads (18). Recently, betaglycan was identified as a co-receptor that
binds inhibin and increases the affinity of inhibin for the type II
activin receptors. When inhibin is bound to betaglycan it also
binds to ActRII and ActRIIB and thereby sequesters them, preventing
formation of the type II/type I receptor complex in response to activin
and, thus, blocking activin signaling. This mechanism elaborates a
model of inhibin function, where inhibin, as a competitive antagonist,
competes with activin for access to ActRII and ActRIIB (19).
In most studies the effects of inhibin have been explained by this
blockade of activin signaling (20, 21), but some inhibin effects have
been reported that seem inconsistent with this mode of action. These
include responses to inhibin in systems that do not respond to activin
(22) and systems that, instead of exhibiting antagonistic interactions,
show similar responses to inhibin and activin (23, 24). These findings
led to the suggestion that inhibin may have additional mechanisms of
action, such as its own independent signaling pathway (25), although
there is little direct functional or biochemical evidence for this
pathway. The role of betaglycan in targeting inhibin to ActRII and
ActRIIB coupled with the involvement of these receptors in BMP
signaling suggested to us that inhibin may antagonize other ligands
that signal through ActRII and ActRIIB. Here we show that inhibin can, indeed, function as an antagonist of BMP responses by competing with
BMPs for binding to type II receptors.
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EXPERIMENTAL PROCEDURES |
Materials--
NuPAGE gels and molecular weight markers were
obtained from Invitrogen. Recombinant human activin-A and inhibin-A
were generated using a stable activin-expressing cell line and were
purified by Wolfgang Fischer (Laboratories for Peptide Biology,
Salk Institute, La Jolla). Recombinant human BMP-2, BMP-7, and
GDF-5 were purchased from R&D systems (Minneapolis, MN), and in
addition BMP-2, BMP-7, and BMP-9 were provided by Genetics Institute
(Boston, MA). 125I-BMP-7 and 125I-inhibin-A
were prepared using the chloramine T method as previously described
(26). Recombinant human TGF
-1, anti-Myc (9E10) monoclonal antibody,
protein G-agarose, and protein A-agarose were purchased from
Calbiochem. Monoclonal anti-FLAG (M2) antibody was purchased from
Sigma. 3,3',5,5'-Tetramethyl benzidine substrate,
bis(sulfosuccinimidyl) suberate (BS3), and chemiluminescent
substrate (SupersignalTM) were purchased from Pierce.
Anti-Smad1 antibody was purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). Affinity-purified, polyclonal rabbit anti-ALK2, anti-ActRII,
and anti-ActRIIB antibodies (directed against residues 474-494 of
mouse ALK2, residues 482-494 of mouse ActRII, and residues 524-536 of
rat ActRIIB) have been previously described (26). Anti-phospho-Smad1
antibody was a kind gift of Peter ten Dijke (The Netherlands Cancer
Institute, Plesmanlaan, The Netherlands). The 4xBRE-Luc,
Myc-betaglycan, and BMPRII-FLAG constructs used in this study were
generously supplied by Joan Massagué (Memorial Sloan-Kettering
Cancer Center, NY).
Transfection and Luciferase Assays in HepG2 Cells--
HepG2
cells were grown at 37 °C in a 5% CO2 humidified
incubator in
-modification of Eagle's medium (Fisher
Mediatech, Pittsburgh, PA) supplemented with 10% fetal bovine serum,
penicillin, streptomycin, and L-glutamine. HepG2 cells were
transfected in 24-well plates (surface area ~1.75 cm2)
with the BRE-Luc reporter plasmid (27), RSV-
-galactosidase, and
either pcDNA3 empty vector or rat betaglycan cDNA with an N-terminal Myc epitope tag (28) in a DNA ratio of 9 µg/1 µg/0.5 µg. Transfections were performed using Superfect Transfection Reagent
(Qiagen, Hilden, Germany) according to the manufacture's recommendations. After 20 h new medium was added, and cells were treated with TGF
1, activin-A, BMP-2, BMP-7, BMP-9, or GDF-5 with or
without inhibin-A as indicated. Sixteen hours after treatment, cells
were harvested in solubilization buffer (1% Triton X-100, 25 mM HEPES, pH 7.8, 15 mM MgSO4, 5 mM EGTA), and luciferase reporter activity was measured and
normalized to relative
-galactosidase activities using standard methods.
Protein Harvest, Electrophoresis, and Immunoblotting--
For
phosphorylation assays HepG2 or TM4 cells growing on 10-cm plates
(surface area ~78 cm2) were treated as shown with BMP-7
and/or inhibin-A for 30 min. Cells were lysed in 1% Triton X-100 in 50 mM Tris, pH 7.5, 50 mM NaCl for 60 min with
gentle rocking at 4 °C. SDS-PAGE was carried out under reducing
conditions on NuPAGE gels (Invitrogen). Electroblotting to
nitrocellulose membranes was carried out in a Invitrogen X-cell II
apparatus according to the manufacturer's instructions. Unbound sites
were blocked at 4 °C with 5% (w/v) skim milk powder in
Tris-buffered saline overnight. Blocked membranes were incubated for
2 h at room temperature with a 1/1000 dilution of a rabbit
polyclonal anti-phospho-Smad1 antibody or a 1/200 dilution of a goat
polyclonal anti-Smad1 (N18) antibody. Membranes were then washed 3 times for 10 min each with Tris-buffered saline/Tween and incubated for
2 h with 2 µg/ml peroxidase-linked anti-rabbit or anti-goat IgG.
Blots were washed 3 times for 10 min each with Tris-buffered saline/Tween, and reactive bands were visualized using the Pierce SupersignalTM ECL detection system.
Cross-linking and Immunoprecipitation of Endogenous Receptors in
HepG2 Cells--
Covalent cross-linking was carried out by incubating
~107 HepG2 cells on 10-cm tissue culture plates (surface
area ~78 cm2) with ~5 × 107 cpm
125I-BMP-7 or 125I-inhibin-A in a total of 5 ml
of tissue culture media for 2 h at room temperature with gentle
rocking. After this incubation, media was aspirated, and the cells were
washed once in 5 ml of ice-cold HDB (12.5 mM Hepes, pH 7.4, 140 mM NaCl, 5 mM KCl). Cells were then
cross-linked in 1 mM bis(sulfosuccinimidyl) suberate (BS3) in HDB and incubated for 30 min at 4 °C.
Cross-linking reactions were quenched by adding 1 ml of 300 mM Tris, pH 7.5, 300 mM NaCl to each plate.
Cells were washed once in 5 ml of HDB and solubilized in 2 ml of lysis
buffer (1% Triton X-100, 50 mM Tris, pH 7.5) for 30 min at
4 °C. Cell material was scraped into Eppendorf tubes, and Triton
X-100-insoluble material was removed by centrifugation at 10,000 rpm
for 5 min. 1 µg of equivalent anti-receptor antibody was added to
each supernatant as shown, and tubes were incubated for 16 h at
4 °C. Immune complexes were precipitated by adding 20 µl of 50%
protein A-agarose slurry to each tube, incubating an additional 1 h at 4 °C, and pelleting the resulting immobilized immune complexes
by centrifugation with 3 washes in 1 ml of lysis buffer. Each
immunoprecipitate pellet was heated at 70 °C for 10 min with or
without dithiothreitol reducing agent and eluted in 50 µl of NuPAGE
SDS sample buffer (Invitrogen) and resolved via SDS-PAGE. SDS-PAGE was
carried out under nonreducing conditions for inhibin-A cross-linking
and under reducing conditions for BMP-7 cross-linking on 3-8% Tris
acetate NuPAGE gels (Invitrogen) in a Invitrogen X-cell II apparatus
according to the manufacturer's instructions. Gels were dried under
vacuum and exposed to film.
Inhibin Binding and Cross-linking in Transfected HEK 293 Cells--
Human embryonic kidney (HEK) 293 cells were grown at
37 °C in a 5% CO2 humidified incubator in DMEM media
(Invitrogen) supplemented with 10% fetal bovine serum, penicillin,
streptomycin, and L-glutamine. HEK 293 cells were
transfected with BMPRII-FLAG (9.5 µg) and Myc-betaglycan (0.5 µg),
adding pcDNA3 to 10 µg of total as shown. Transfections were
performed using the GenePorter 2 reagent (Gene Therapy Systems, San
Diego, CA) according to the manufacture's recommendations. For binding
assays transfection was performed using ~1 × 105
HEK 293 cells/well in 24-cell plates (surface area ~1.75
cm2) with 0.5 µg of DNA/well. For cross-linking assays
~2 × 106 HEK 293 cells/10-cm dish (surface area
~78 cm2) and 10 µg of DNA were used. After incubating
overnight, transfection reagent and DNA-containing media were removed,
and cells were washed and allowed to recover for 24 h in 10% FBS,
DMEM. Binding assays were performed in triplicate wells using ~2 × 106 cpm 125I-inhibin-A/well for 2 h at
room temperature in 400 µl of DMEM containing 1% BSA. Wells were
washed 3 times in HDB containing 1% BSA, and bound counts were
solubilized in 1% SDS and counted in a
counter. For cross-linking,
~1 × 107 cpm 125I-inhibin-A was
incubated in 5 ml of DMEM, 10% FBS for 2 h at room temperature
with gentle rocking. As described above for HepG2 cross-linking, cells
were washed and cross-linked in 1 mM bis(sulfosuccinimidyl) suberate (BS3) for 30 min at 4 °C. Cross-linking was
quenched, and cells were washed and solubilized in 2 ml of lysis buffer
for 30 min at 4 °C. Cell material was scraped into Eppendorf tubes,
and Triton X-100-insoluble material was removed by centrifugation at
10,000 rpm for 5 min. 10 µg of anti-FLAG (M2, Sigma) or 2 µg
anti-Myc antibody (9E10, Calbiochem) was added to each supernatant and incubated for 16 h at 4 °C. Immune complexes were precipitated by adding 20 µl of 50% protein G-agarose slurry, incubating for 1 h at 4 °C, and pelleting the resulting immobilized immune
complexes by centrifugation with 3 washes in 1 ml of lysis buffer.
Pellets were heated at 70 °C for 10 min in 50 µl and resolved via
SDS-PAGE on 3-8% Tris acetate NuPAGE gels.
Generation of Stable C2C12 Cell Lines and
Alkaline Phosphatase Assays--
C2C12 cells
were grown at 37 °C in a 5% CO2 humidified incubator in
DMEM media (Invitrogen) supplemented with 10% fetal bovine serum,
penicillin, streptomycin, and L-glutamine.
C2C12 cells were transfected with a retroviral
construct containing the neomycin cassette and the rat betaglycan
cDNA containing an N-terminal Myc epitope tag. After 2 days single
cells were isolated by dilution cloning in media containing 400 µg/ml
G418. Clones were amplified, and cell surface betaglycan expression was
measured by cell surface enzyme-linked immunosorbent assay (26).
Briefly, stable C2C12 cell clones were screened
in triplicate in 96-cell plates for Myc-betaglycan expression. Each
well was rinsed with HDB, and cells were fixed in 4% paraformaldehyde
for 30 min at 4 °C. Cells were then rinsed with HDB, blocked with
3% BSA in HDB for 30 min at room temperature, rinsed with HDB, and
incubated for 2 h with 2 µg/ml anti-Myc antibody in 3% BSA in
HDB. Cells were then rinsed 3 times with HDB and incubated with
peroxidase-conjugated anti-mouse IgG in 3% BSA in HDB for 1 h at
room temperature. Wells were rinsed 3 times with HDB, 100 µl of
3,3',5,5'-tetramethyl benzidine peroxidase substrate (Pierce)
was added to each well, and plates were incubated at room temperature
until color was visible. Reactions were stopped by adding 100 µl of
0.18 M H2SO4 to each well, and
peroxidase activity was quantified by measuring the absorbance of the
resulting yellow solutions at 405 nm. Some Myc-betaglycan-expressing
stable cell lines were screened for inhibin blockade of BMP-2-induced alkaline phosphatase activity. Alkaline phosphatase activity was measured using standard methods (29). Briefly,
C2C12 cells were plated at 2.5 × 104 cells/well in 96-well plates in 50 µl of
differentiation media (DMEM with 1% FBS with penicillin, streptomycin,
and L-glutamine). Two hours later wells were treated with
differentiation media containing BMP-2 and/or inhibin-A as shown to a
total volume of 100 µl/well. Cells were allowed to differentiate for
4 days at 37 °C in a 5% CO2 humidified incubator. For
quantitative alkaline phosphatase assays, triplicate wells were washed
once in HDB and lysed for 60 min in 100 µl of 1% Nonidet P-40, 100 mM glycine, pH 9.6, 1 mM MgCl2, 1 mM ZnCl2. To measure alkaline phosphatase activity cell lysates were incubated with 100 µl of 1 mg/ml
p-nitrophenyl phosphate in 100 mM glycine, pH
9.6, 1 mM MgCl2, 1 mM
ZnCl2 until color developed, and activity was measured by
absorbance at 405 nm. Pictures of differentiated myotubes were taken in
low magnification (20×), bright field conditions without fixation
using a Canon EOS Elan II camera.
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RESULTS |
Inhibin Antagonism of BMP Responses in HepG2 and TM4 Cells--
To
investigate if inhibin can antagonize BMP signaling we first tested if
inhibin-A could block BMP responses in HepG2 cells. The hepatocyte
HepG2 cell line was used as a model BMP-responsive system because BMPs
are expressed in the liver and are proposed to be involved in local
liver homeostasis (30) as well as in liver development (31). HepG2
cells express both type I and type II BMP receptors and are
BMP-responsive (30). As shown in Fig. 1,
BMP-2, BMP-7, BMP-9, and to a much lesser extent GDF-5 stimulated
BRE-Luc, a BMP-responsive promoter construct (27), in HepG2 cells.
Activin-A did not stimulate BRE-Luc (Fig. 1) but did stimulate an
activin-responsive reporter in the same cells (32), indicating that
BRE-Luc activation is BMP-specific. Activin responses in HepG2 cells,
including promoter induction and cell growth (33), were blocked by
inhibin-A. Although inhibin-A did not affect BRE-Luc activity when
added alone, BRE-Luc stimulation by BMP-2, BMP-7, BMP-9, and GDF-5 was
blocked by inhibin-A when both factors were added together (Fig. 1),
clearly indicating that inhibin can antagonize BMP responses.

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Fig. 1.
Inhibin antagonizes BMP promoter
induction. HepG2 cells transfected with BRE-Luc and RSV- Gal
were treated with 200 pM TGF -1, 10 nM
inhibin-A, or 2 nM activin-A, BMP-2, BMP-7, BMP-9, or GDF-5
as shown, harvested, and assayed as described under "Experimental
Procedures." Luciferase activity is shown in arbitrary luciferase
units, normalized to -galactosidase activity.
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To test if inhibin antagonism of BMP induction of BRE-Luc activity was
mirrored in a blockade of BMP signaling, we examined Smad1
phosphorylation. BMP signaling is mediated by Smad1, which is activated
by type I receptor phosphorylation of a C-terminal SSXS
motif. Immunoblotting HepG2 cell lysates with an antibody directed
against the phosphorylated form of Smad1 revealed that the rapid,
30-min induction of Smad1 phosphorylation by BMP-2 was blocked by
inhibin-A (Fig. 2A). Inhibin-A
did not induce Smad1 phosphorylation, and total levels of Smad1 in the
cells were not affected by either treatment. Inhibin antagonism of BMP
transcriptional responses are, therefore, probably not specific for the
BRE reporter but, rather, reflect a general block of BMP signaling in
inhibin-treated HepG2 cells.

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Fig. 2.
Inhibin antagonizes BMP induced Smad1
phosphorylation. HepG2 cells were treated with 2 nM
BMP-2 (A) or TM4 cells were treated with 2 nM
BMP-7 (B) with or without 10 nM inhibin-A for 30 min, harvested, and subjected to Western blot analysis as described
under "Experimental Procedures." Smad1 was detected using
polyclonal antibodies directed against total Smad1 or phosphorylated
Smad1. Molecular weight markers are represented as
Mr × 10 3.
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To examine if this loss of Smad signaling was a general phenomenon we
attempted to determine if inhibin could prevent
BMP-dependent Smad phosphorylation in other BMP-responsive
cell types. The physiological role of BMPs in the adult includes
regulation of spermatogenesis in the testis (7), where inhibin is also
highly expressed. Therefore, we selected the mouse Sertoli TM4 cell
line, which expresses betaglycan, to examine if inhibin could prevent
BMP-induced Smad signaling. Phospho-Smad1 immunoblotting revealed that
30 min of BMP-7 treatment induced Smad1 phosphorylation in TM4 cells (Fig. 2B). This BMP-7 effect was blocked by co-treatment
with inhibin-A. Similar to HepG2 cells, inhibin-A did not induce Smad1 phosphorylation or alter total levels of Smad1, suggesting this is a
specific block of BMP signaling. These results demonstrated that
inhibin-A could function as an antagonist of BMP signaling in multiple
BMP-responsive cell types. The fact that inhibin-A blocked rapid BMP
effects immediately downstream of the BMP receptor complex suggested
that inhibin antagonism is most probably direct, likely occurring
through disruption of the BMP receptor complex.
Betaglycan Potentiates Inhibin Antagonism of BMP
Signaling--
BMP and inhibin effects on BRE-Luc activity were
concentration-dependent. As shown in Fig.
3A, BMP-7 stimulated BRE-Luc
activity in a dose-dependent manner with a maximal effect
at 1-2 nM. The concentration-dependent
inhibitory effect of inhibin-A on BRE-Luc activation by 1 nM BMP-7 was near maximum at 10 nM inhibin-A. These results indicated that inhibin could completely antagonize signaling by maximal doses of BMP-7. Stimulation of BRE-Luc activity by
BMP-7 and its antagonism by inhibin-A occurred at physiologically relevant concentrations, showing potencies similar to those seen in
other responsive systems.

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Fig. 3.
Inhibin and BMPs have
dose-dependent effects mediated by betaglycan.
A, HepG2 cells transfected with BRE-Luc and RSV- Gal were
treated with increasing doses of BMP-7 as shown, harvested, and assayed
as described under "Experimental Procedures." B, HepG2
cells were transfected with BRE-Luc, RSV- Gal, and Myc-betaglycan
(BG) or empty vector (pcDNA3) as shown. Cells
were treated with 1 nM BMP-7 and increasing doses of
inhibin-A, harvested, and assayed as described under "Experimental
Procedures."
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We next examined whether the inhibin suppression of BMP signaling would
be facilitated by the presence of betaglycan, as is the case for the
abrogation of activin signaling by inhibin. HepG2 cells endogenously
express betaglycan, and as with other cells, transfection of more
betaglycan decreased the IC50 of inhibin-A for antagonism
of activin-A (data not shown). Similarly, betaglycan transfection
increased the potency of inhibin-A blockade of BRE-Luc induction by
BMP-7 (Fig. 3B). Betaglycan transfection lowered the
IC50 of inhibin-A from ~100 pM to less than 1 pM. Both the magnitude of the shift and the final potency
are similar to the values observed for antagonism of activin signaling
by inhibin in these and other cells (19). These data demonstrate that
transfected betaglycan can enhance inhibin antagonism of BMP responses,
consistent with the idea that inhibin disrupts the BMP receptor complex.
Inhibin-A and BMP-7 Compete for Type II Activin
Receptors--
HepG2 cells endogenously express the BMP receptors
ActRII, ActRIIB, and ALK-2. To investigate the mechanism of inhibin
antagonism of BMP signaling we tested for competition between inhibin-A
and BMPs for binding to the type II activin receptors. Inhibin competed for activin binding to ActRII and ActRIIB in HepG2 cells. Because we
could not reproducibly measure 125I-BMP-7 binding due to
high levels of nonspecific binding (data not shown), we utilized
covalent cross-linking to visualize inhibin-A or BMP-7 bound to ActRII
and ActRIIB. As shown in Fig.
4A, a cross-linked complex of
125I-inhibin-A bound to betaglycan and ActRII (lane
1) or ActRIIB (lane 5) could be immunoprecipitated from
HepG2 cells. Unlabeled inhibin-A completely competed with
125I-inhibin-A for these complexes (lanes 3 and
7), but unlabeled BMP-7 was only partially effective
(lanes 2 and 6). Unlabeled BMP-7 competed with
the inhibin-betaglycan complex for type II receptors and, thus,
decreased the amount of 125I-inhibin-A and betaglycan
isolated in type II receptor immunoprecipitates.

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Fig. 4.
Inhibin and BMPs compete for binding type II
activin receptors. A, 125I-inhibin-A was
bound and cross-linked to HepG2 cells in the presence of unlabeled
BMP-7 or inhibin-A as indicated, cells were lysed, and cross-linked
complexes were purified by immunoprecipitation, as described under
"Experimental Procedures." BG, Myc-betaglycan.
B, 125I-BMP-7 was bound and cross-linked to
HepG2 cells in the presence of unlabeled BMP-7 or inhibin-A as
indicated, then cells were lysed, and cross-linked complexes were
purified by immunoprecipitation, as described under "Experimental
Procedures." The position of the molecular weight markers is shown,
and arrows indicate cross-linked complexes.
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Cross-linking and immunoprecipitation using 125I-BMP-7
confirmed that inhibin-A and BMP-7 competed for type II receptor
binding. Different BMPs selectively bind to different combinations of
type I and type II receptors. In the presence of the type I receptor, ALK-2, BMP-7 can bind to ActRII, ActRIIB, and BMPRII. As shown in Fig.
4B, 125I-BMP-7-cross-linked complexes could be
immunoprecipitated from HepG2 cells using antibodies to ActRII
(lane 1), ActRIIB (lane 5), and ALK-2 (lane
8). Unlabeled BMP-7 competed with 125I-BMP-7 for
binding to these complexes (lane 3, 7, or
10, respectively). Unlabeled inhibin-A also competed with
125I-BMP-7 (lane 2, 6, or
9), confirming that inhibin-A can displace BMP-7 binding in
HepG2 cells. Similar results were seen using 125I-BMP-2
(data not shown). BMP-7 and inhibin-A binding to ActRII and ActRIIB
could also be competed with activin-A in HepG2 cells, similar to other
cell types (34). Thus, inhibin competes with BMP-7 for crucial type II
receptor binding sites. This ability of inhibin to prevent BMP binding
to ActRII and ActRIIB could explain the functional antagonism of BMP
signaling by inhibin we observed in HepG2 and other cell types. If BMPs
are prevented from assembling a type I-type II receptor complex, this
would prevent all BMP signaling in target cells.
Betaglycan Allows Inhibin to Bind to BMPRII--
A model in which
inhibin and BMP compete for type II receptors raises important
questions concerning BMPRII. BMPs can utilize ActRII, ActRIIB, and
BMPRII, but inhibin had only been shown to block activin binding to
ActRII and ActRIIB. Whether inhibin would disrupt BMP signaling through
BMPRII or not was unclear. When expressed alone BMPRII did not bind
activin (12), but inhibin binding had not been investigated. To resolve
this question, we examined 125I-inhibin-A binding in HEK
293 cells transfected with epitope-tagged BMPRII and betaglycan. When
expressed alone BMPRII did not bind 125I-inhibin-A (Fig.
5A). However, co-expression of
BMPRII with betaglycan resulted in ~10-fold more
125I-inhibin-A binding than expression of betaglycan alone
(Fig. 5A). This effect was not due to changes in betaglycan
expression levels and is similar to the synergistic binding of inhibin
to ActRII and betaglycan but differs in that ActRII binds inhibin in the absence of betaglycan (19). Another difference between ActRII
and BMPRII binding to inhibin emerges when the binding data are
normalized to determine binding affinities. As shown in Fig.
5B, the affinity of inhibin for HEK 293 cells co-expressing betaglycan and BMPRII (~900 pM) was actually lower than
for cells expressing betaglycan alone (~530 pM). This
contrasts sharply with the pattern seen in cells expressing betaglycan
and/or ActRII. Cells co-expressing ActRII and betaglycan have a 3-fold
higher affinity for inhibin (~200 pM) than cells
expressing betaglycan alone (~600 pM) and a 30-fold
higher affinity than cells expressing ActRII alone (~6
nM) (19). This suggests that betaglycan confers on BMPRII
the ability to bind inhibin but, unlike ActRII or ActRIIB, BMPRII does
not improve the affinity of inhibin for the complex.

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Fig. 5.
Betaglycan allows inhibin to bind to BMPRII.
A, HEK 293 cells were transfected with empty vector
(pcDNA3), BMPRII-FLAG (BRII), Myc-betaglycan
(BG), or BMPRII-FLAG and Myc-betaglycan
(BRII+BG), as indicated, and competition binding assays were
performed with unlabeled inhibin-A as described under "Experimental
Procedures." Data are presented as 125I-inhibin-A bound
as counts/min. B, the Myc-betaglycan (BG) or
BMPRII-FLAG and Myc-betaglycan (BRII+BG) data in
A, normalized and presented as percent specific binding.
C, HEK 293 cells were transfected with pcDNA3 vector
(vector), BMPRII-FLAG (BRII), Myc-betaglycan
(BG), or BMPRII-FLAG and Myc-betaglycan (BRII+BG)
as indicated, bound with 125I-inhibin-A, cross-linked, and
purified by immunoprecipitation (IP) as described under
"Experimental Procedures." The positions of molecular weight
markers are represented as Mr × 10 3, and cross-linked complexes are shown with
arrows.
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To confirm that the increase in inhibin binding was due to BMPRII
participation in the inhibin-betaglycan complex, we determined if
BMPRII could interact with betaglycan and directly bind inhibin. We
performed cross-linking experiments with 125I-inhibin-A
followed by immunoprecipitation using antibodies against the FLAG and
Myc epitope tags on the transfected receptors. Immunoprecipitated complexes from HEK 293 cells transfected with vector or BMPRII did not
contain detectable 125I-inhibin-A-cross-linked complexes
(Fig. 5C). Cells transfected with betaglycan contained low
but detectable levels of betaglycan cross-linked to inhibin-A
(lane 3). High levels of
125I-inhibin-A-cross-linked complexes could be
immunoprecipitated from cells co-transfected with BMPRII and
betaglycan. Unlabeled inhibin-A can compete for both BMPRII (lane
4) and betaglycan (lane 6) binding. These complexes
could be immunoprecipitated with antibodies to the epitope tags on
either BMPRII (lanes 4 and 5) or betaglycan
(lanes 6 and 7), demonstrating that inhibin-A interacted with both BMPRII and betaglycan simultaneously.
Betaglycan Expression Causes Cells to Become Sensitive to
Inhibin--
We sought to identify BMP-responsive cells that are
insensitive to inhibin to test if betaglycan could confer inhibin
sensitivity on otherwise insensitive cells. We tested BMP responses in
the mouse C2C12 myoblast precursor line and
found they were poorly antagonized by inhibin. Under low serum
conditions C2C12 cells differentiate to a
myotube-like phenotype; BMP-2 shifts differentiation to an
osteoblast-like cell expressing alkaline phosphatase (29). As shown in
Fig. 6A, inhibin did not
antagonize BMP-2 induction of alkaline phosphatase activity. Even
extremely high inhibin concentrations of 100 nM or greater
produced less than 20% inhibition (data not shown). However, in
C2C12 cells stably transfected with betaglycan,
BMP-2 responses are sensitive to inhibin-A (Fig. 6A, right), similar to the effects betaglycan expression had on
inhibin antagonism of activin signaling in AtT20 cells (19). Stable expression of betaglycan also caused profound changes in the ability of
inhibin to antagonize the effects of BMP-2 on myotube formation. BMP-2
treatment blocked myotube differentiation (Fig. 6B, compare top to middle). Stable expression of betaglycan
did not alter the effect of BMP-2 on myotube formation in
C2C12 cells (Fig. 6B) or on
BMP-2-induced alkaline phosphatase activity (Fig. 6A). Inhibin alone had no effect on myotube formation in parental or betaglycan stable C2C12 cells (data not shown).
However, the effect of BMP-2 in C2C12 cells
stably expressing betaglycan could be completely blocked by inhibin-A
(Fig. 6B, bottom right), whereas the response to
BMP-2 in parental C2C12 cells was completely
resistant to inhibin-A (Fig. 6B, bottom left).
These data established that in C2C12 cells
betaglycan could be a key effector of inhibin antagonism of BMP
signaling, and thus, betaglycan could play a crucial role in
establishing the sensitivity to inhibin of both activin and BMP
responses. This implies that inhibin cannot function as a BMP
antagonist under all conditions but, rather, is only effective in the
appropriate cellular context.

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Fig. 6.
Betaglycan renders
C2C12 cells sensitive to inhibin antagonism.
A, parental C2C12
(C2C12) or
C2C12 stably expressing Myc-betaglycan
(BG-C2C12) were treated with 10 nM BMP-2 or 5 nM inhibin-A as shown, grown for
4 days, and lysed, and alkaline phosphatase activity was measured as
described under "Experimental Procedures." B, parental
C2C12 or BG-C2C12 cells
were treated as above. Cell morphology under low magnification (20×),
bright field photography reveals cells have fused to myotubes
(a, b, f) or are a monolayer of
osteoblast-like cells (c, d, e).
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DISCUSSION |
The data presented here demonstrate that inhibin can block BMP
responses and reveal a novel function of inhibin as a potentially important physiological antagonist of BMPs. Inhibin-A potently blocks
phosphorylation of Smad1 induced by BMPs and BMP induction of BRE-Luc.
Inhibin-A blocks BMP-7 binding to endogenous type II receptors in HepG2
cells. As is the case for activin, the inhibin antagonism of BMP
responses is potentiated by betaglycan. Betaglycan also supports
inhibin binding to BMPRII. This dependence on betaglycan to allow
inhibin binding to BMPRII suggests that inhibin is even more dependent
on betaglycan for the blockade of BMP responses than inhibin is for the
blockade of activin responses. The importance of betaglycan in the
inhibin blockade of BMP responses is further demonstrated in
C2C12 cells by the absence of inhibin effects on BMP-2 responses unless betaglycan is expressed.
These data support a model in which inhibin functions as a direct
competitive antagonist of BMP binding to type II receptors. In this
model inhibin binds to and sequesters type II receptors and, thus,
prevents BMPs from initiating signaling in target cells. In this model
inhibin appears to antagonize BMP signaling through a mechanism similar
to that proposed for inhibin antagonism of activin signaling. Thus, we
would predict inhibin should antagonize responses to all BMPs in a
target cell irrespective of the BMP receptors present if
betaglycan or a functionally similar co-receptor is expressed. In
cells that do not express betaglycan or similar molecules we would
predict inhibin would block BMP responses poorly if at all. With the
findings presented here betaglycan has now been implicated in the
regulation of signaling by BMPs in addition to its roles with TGF
s,
activins, and inhibins. The binding of inhibin to BMPRII only in the
presence of betaglycan is analogous to the effect of betaglycan on
TGF
2 binding to T
RII, suggesting similarities in betaglycan
binding and type II receptor interaction with diverse TGF
superfamily members. Overall, the data suggest that inhibin may not be
a specific antagonist of activin signals but, instead, a more general
antagonist of TGF
superfamily signaling.
This ability of inhibin to block BMP responses may explain the inhibin
effects attributed to independent inhibin signaling pathways (25).
These effects have seemed inconsistent with the model of inhibin,
acting in cooperation with betaglycan as an activin receptor
antagonist. Based on our data the reports of inhibin effects in systems
that do not respond to activin may well be due to inhibin antagonizing
the well documented BMP actions in these systems. For example, inhibin
was reported to block BMP-induced differentiation in primary bone cell
cultures that did not respond to activin, and the activin antagonist
follistatin did not mimic inhibin effects (22). Whether these results
could be explained as an inhibin antagonism of the BMP-mediated
differentiation in these cells should be tested. This wider inhibin
specificity may explain inhibin actions without invoking a putative,
currently unknown intracellular signaling pathway activated by inhibin, although our data do not exclude this possibility. Given the fact that
activin and BMP signaling have distinct and often opposing actions
(35), inhibin antagonism of BMP responses may also explain reports that
activin and inhibin have similar effects in some systems (23, 24), a
hypothesis that should be tested in future studies.
The inhibin blockade of BMP responses expands the already extensive
regulatory network that limits BMP effects. Signaling by the BMP family
is regulated by a diverse group of extracellular binding proteins that
control BMP availability. BMP-binding proteins, including noggin (36),
gremlin (37), chordin (38), cerberus (39, 40), DAN (41), and
follistatin (42) selectively bind to subsets of BMPs and regulate BMP
activity by preventing BMP binding to cell surface receptors (43).
Binding protein specificity is based on the availability of the binding
protein and the selectivity and affinity of the binding protein for its
target ligands. We have shown that inhibins also behave as BMP
antagonists. Unlike binding proteins, inhibin selectivity is defined by
betaglycan expression on target cells. Therefore, the specificity of
inhibin antagonism depends not only on inhibin availability but also on betaglycan expression in target cells, potentially allowing for selective blockade of a subset of BMP responses in a target organ with
multiple cell types. Based on this model we predict that cells that do
not express betaglycan would be insensitive to inhibin.
Inhibin antagonism of BMP responses may still be relatively widespread,
given that betaglycan is broadly, although not universally, expressed
(44). Although BMPs were first recognized as proteins involved in bone
formation, it is now clear that they have roles throughout development
and during adult life, including many completely unconnected to bone
morphogenesis. These include the regulation of reproductive function,
specifically the differentiation and development processes involved in
both spermatogenesis and oogenesis (6, 7, 45-50). Although the roles
of inhibin in development are poorly defined, inhibin is clearly an
important regulator of reproductive function in the adult (3, 51-54).
BMPs and inhibins are both expressed and have functional roles in the
gonads, suggesting interactions between inhibin and BMPs are likely to
be physiologically important. This idea is further supported by a
report that BMP-2 induces inhibin-B in granulosa cells (51). Clearly,
examining inhibin-BMP interactions in these physiological systems will
be important areas of future research. Based on our findings presented here we suggest that inhibin may serve to restrict and refine the BMP
responsiveness of different cell populations. Inhibin and betaglycan
serve to narrow the range of cells that can sense broadly acting TGF
superfamily ligands in order to limit ligand effects to select target
cells. Our findings suggests inhibins, in concert with betaglycan or a
functionally similar co-receptor, may play more widespread and
important roles in regulating cell to cell communication than
previously understood.