Inhibin Is an Antagonist of Bone Morphogenetic Protein Signaling*

Ezra WiaterDagger and Wylie Vale§

From the Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, California 92037 and Dagger  Department of Biology Graduate Program, University of California, San Diego, La Jolla, California 92093

Received for publication, September 21, 2002, and in revised form, December 9, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Inhibins are endogenous antagonists of activin signaling, long recognized as important regulators of gonadal function and pituitary FSH release. Inhibin, in concert with its co-receptor, betaglycan, can compete with activin for binding to type II activin receptors and, thus, prevent activin signaling. Because bone morphogenetic proteins (BMPs) also utilize type II activin receptors, we hypothesized that BMP signaling might also be sensitive to inhibin blockade. Here we show that inhibin blocks cellular responses to diverse BMP family members in a variety of BMP-responsive cell types. Inhibin abrogates BMP-induced Smad signaling and transcription responses. Inhibin competes with BMPs for type II activin receptors, and this competition is facilitated by betaglycan. Betaglycan also enables inhibin to bind to and compete with BMPs for binding to the BMP-specific type II receptor BMPRII, which does not bind inhibin in the absence of betaglycan. Betaglycan can confer inhibin responsiveness on cells that are otherwise insensitive to inhibin. These findings demonstrate that inhibin, acting through betaglycan, can function as an antagonist of BMP responses, suggesting a broader role for inhibin and betaglycan in restricting and refining a wide spectrum of transforming growth factor beta  superfamily signals.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Activins, inhibins, and BMPs1 are structurally related members of the TGFbeta 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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 TGFbeta -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 alpha -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-beta -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 TGFbeta 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 beta -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 gamma  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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta Gal were treated with 200 pM TGFbeta -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 beta -galactosidase activity.

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.

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-beta 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-beta 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."

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.

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.

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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 TGFbeta s, activins, and inhibins. The binding of inhibin to BMPRII only in the presence of betaglycan is analogous to the effect of betaglycan on TGFbeta 2 binding to Tbeta RII, suggesting similarities in betaglycan binding and type II receptor interaction with diverse TGFbeta superfamily members. Overall, the data suggest that inhibin may not be a specific antagonist of activin signals but, instead, a more general antagonist of TGFbeta 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 TGFbeta 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.

    ACKNOWLEDGEMENTS

We thank Louise Bilezikjian, Peter Gray, Craig Harrison, and Senyon Choe for providing helpful discussions. BMP-2, BMP-7, and BMP-9 were provided by Genetics Institute. Inhibin-A was provided by Wolfgang Fisher (Salk Institute). Anti-phospho-Smad1 antibody was generously provided by Peter ten Dijke. The BRE-Luc, BMPRII-FLAG, and Myc-betaglycan constructs were kindly provided by Joan Massagué.

    FOOTNOTES

* This work was supported by National Institutes of Health Program Project Grant HD13527 and by The Kleberg Foundation and the Foundation for Medical Research, Inc.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.

§ An FMR, Inc., Senior Investigator. To whom correspondence should be addressed. Fax: 858-552-1546; E-mail: vale@salk.edu.

Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M209710200

    ABBREVIATIONS

The abbreviations used are: BMP, bone morphogenetic protein; TGF, transforming growth factor; HDB, Hepes dissociation buffer; HEK cells, human embryonic kidney cells; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; GDF, growth and differentiation factor.

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