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Molecular Endocrinology 15 (4): 668-679
Copyright © 2001 by The Endocrine Society

Modulation of Activin Signal Transduction by Inhibin B and Inhibin-Binding Protein (InhBP)

Stacey C. Chapman and Teresa K. Woodruff

Department of Neurobiology and Physiology (S.C.C., T.K.W.) Northwestern University and Department of Medicine (T.K.W.) Northwestern University Medical School Evanston, Illinois 60208-2850


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
An antagonistic relationship between inhibin and activin is essential to the control of pituitary FSH release and to normal gonadal function. Two inhibin ligands, inhibin A and inhibin B, are made by the ovary in females, and each regulate pituitary FSH at different times during the reproductive cycle. Inhibin B, but not inhibin A, is produced by the testes and is therefore responsible for all inhibin-dependent FSH regulation in males. Although the activin signal transduction pathway has been well characterized, little is known about the mechanism of inhibin signaling and its relationship to activin antagonism. A recently cloned inhibin-binding protein, InhBP (p120), associates strongly with the type IB activin receptor (Alk4) in a ligand-responsive manner and interacts to a lesser extent with other activin and bone morphogenetic protein (BMP) type I and activin type II receptors. Activin stimulates the association of InhBP and Alk4, and inhibin B, but not inhibin A, interferes with InhBP-Alk4 complex formation. InhBP is necessary to mediate a specific antagonistic effect of inhibin B on activin-stimulated transcription. Appropriate stoichiometry between InhBP and the activin type I receptor is crucial to InhBP function. These findings suggest that InhBP is an inhibin B-specific receptor that mediates antagonism of activin signal transduction through the modulation of activin heteromeric receptor complex assembly.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hormones and growth factors produced by the hypothalamus, pituitary, and gonads control normal reproductive function. The pituitary hormones FSH and LH are produced by gonadotrope cells in the pituitary (reviewed in Ref. 1), and release of these hormones is facilitated by the hypothalamic releasing peptide, GnRH (reviewed in Ref. 2). FSH and LH regulate ovarian follicle maturation and ovulation in the female and maintain tonic testicular sperm and steroid production in males. Hormones produced by the gonads feed back to the pituitary gonadotrope and hypothalamus to regulate FSH and LH synthesis and secretion. The gonadotropins share a common {alpha}-subunit, yet the synthesis and release of each hormone is often discordant (3), and the precise molecular mechanisms by which LH and FSH are differentially regulated have not been determined. The gonadal hormone inhibin is one of the ligands that mediate differential LH and FSH production. Inhibin is an endocrine hormone that specifically inhibits FSH release in a cycle-dependent manner in females and is critical to normal testicular-pituitary function in males (4, 5). Despite the central nature of gonadal inhibin to the regulation of the reproductive axis, little is known regarding its molecular mechanism of action, largely because a receptor for this ligand had not been identified until recently.

Both inhibin and activin are dimeric hormones and members of the transforming growth factor-ß (TGFß) superfamily of proteins (reviewed in Ref. 6). Inhibin is assigned to the TGFß superfamily because of its ß-subunit, yet it is unique among the ligands because it is capable of heteromeric assembly (7). The inhibin heterodimer consists of an {alpha}-subunit and one of two ß-subunits, ßA (inhibin A) or ßB (inhibin B), and production of this hormone is largely restricted to the ovary and testes (8). Activin is a dimer of ß-subunits produced by many tissues throughout the body and is a local regulator of pleiotropic cell homeostasis (9). In contrast, inhibin is one of the only TGFß superfamily ligands that acts as an endocrine hormone, and known inhibin activity is restricted to a discrete population of cells in the pituitary and the gonads.

Although gonadal-derived inhibin has been primarily regarded as an endocrine agent involved in the regulation of FSH release from the pituitary, specific binding sites have been localized to the ovary and testes of the rat (10). Injection of recombinant human (rh-) inhibin A into the ovarian intrabursal space of female rats results in the growth and accumulation of intermediate (350–500 nm diameter) recruited follicles (11). Furthermore, inhibin A treatment of cultured gonadal cells has been shown to stimulate steroidogenesis in vitro. Production and secretion of androstenedione and dehydroepiandrosterone by cultured primary human thecal cells increase significantly after treatment with rh- inhibin A (12, 13), and rh-inhibin A stimulates expression of the steroidogenic enzyme cytochrome p450-c17 in primary cultures of porcine Leydig cells (14, 15). These findings suggest that inhibin is an endocrine, paracrine, and autocrine hormone of the reproductive axis.

Many, but not all, activin actions are opposed by inhibin (16). Inhibin has been shown to antagonize activin in a variety of physiological circumstances, the best characterized of which is its effect on pituitary FSH production (4). Inhibin also abrogates local activin actions in the gonads. For instance, inhibin has been shown to antagonize activin inhibition of testosterone production in cultured rat Leydig cells and activin stimulated 3ß-hydroxysteroid dehydrogenase (3ß-HSD) expression in cultured porcine Leydig cells (14, 15). The mechanism by which inhibin is able to antagonize activin action is an important aspect of inhibin biology and is most likely multifactorial. Molecular antagonism of activin by inhibin occurs, at least in part, through competition for the inhibin ß-subunit. In the ovary, levels of the {alpha}-subunit far exceed those of the ß- subunit, thus favoring inhibin rather than activin dimer assembly. Inhibin may also block activin action through an interaction with the activin type II receptor (ActRII) (17). The affinity of inhibin for ActRII is 10-fold lower than activin; therefore, an excess of inhibin is required to block activin action through its receptor (18, 19, 20).

In many physiological settings, molecular assembly and receptor competition can not fully account for the antagonistic actions of inhibin and activin. Indeed, many of activin’s actions are insensitive to inhibin antagonism. For example, inhibin cannot antagonize activin-stimulated hemoglobin synthesis in erythrocytes (18), and inhibin does not affect activin-induced granulosa cell growth (21). Thus, a distinct inhibin receptor or other inhibin-binding accessory molecule is necessary to potentiate an inhibin response. Moreover, when all pituitary activin is neutralized by an activin-specific antibody, inhibin is still able to block FSH release, suggesting that not all of inhibin’s actions are a result of activin antagonism (22). Further, an independent inhibin-signaling pathway is predicted based on the ability of inhibin to stimulate hCG-supported testosterone secretion in porcine Leydig cells and steroidogenesis in gonadal tumor cells (12, 13, 15, 23).

Early efforts to purify an inhibin receptor focused on the identification of candidate proteins based on homology to the known receptors of the TGFß superfamily. The cellular response to activin and most other TGFß superfamily members is transduced through a heteromeric receptor complex comprised of two single membrane-spanning serine-threonine kinase subunits (24). Ligand binds to a specific type II receptor, which then phosphorylates and activates a type I receptor. Together, type I and type II receptors form a complex that is then capable of activating downstream signaling events (25, 26). When inhibin binding to type II or type I-like receptors could not be identified in tissues that were clearly sites of inhibin action, affinity purification methodology was used to isolate inhibin-binding proteins from gonadal tumors and bovine pituitaries (27, 28). These efforts led to the cloning of a novel inhibin-binding protein, InhBP, that is the focus of the current investigation.

InhBP, previously called p120, is expressed in inhibin target tissues and is a single membrane-spanning protein that contains 12 Ig domain repeats separated into 5 and 7 repeats by a short linker region (28). InhBP has no discernible intracellular kinase domain, and this predicts a mechanism of inhibin signal transduction that is distinct from the heteromeric receptor model of other members of the superfamily. The following studies describe the characterization of the biophysical and signal-transducing properties of InhBP and address the critical role InhBP plays in mediating inhibin action.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Localization and Inhibin Binding Properties of InhBP
InhBP must be expressed at the cell membrane and bind inhibin to function as an inhibin receptor. The subcellular localization of InhBP was determined by immunofluorescence analysis of CV-1 monkey kidney cells transfected with Flag-tagged InhBP (Fig. 1AGo). InhBP is excluded from the nucleus and is expressed in a diffuse punctate pattern indicative of membrane-localized proteins. Three-dimensional reconstruction of CV-1 cells transfected with InhBP confirmed membrane localization of the expressed, fluorescently tagged construct (data not shown). Because CV-1 cells do not express endogenous InhBP, the ability of InhBP to confer inhibin binding to these cells was determined. CV-1 cells were transiently transfected with Flag-tagged InhBP (Fig. 1Go, C, E, G, and I) and treated with either iodinated inhibin A (Fig. 1Go, B–E) or inhibin B (Fig. 1Go, F–I). InhBP expression was colocalized with inhibin binding by immunostaining with a Flag epitope-directed antibody (Fig. 1Go, B, C, F, and G). Neither iodinated inhibin A nor iodinated inhibin B binds CV-1 cells that have not been transfected with InhBP (Fig. 1Go, B, D, F, and H). The expression of InhBP in CV-1 cells is necessary and sufficient to confer inhibin binding ability to these cells (Fig. 1Go, C, E, G, and I). Thus, InhBP exhibits a subcellular localization pattern and inhibin binding properties that support its role as an inhibin receptor.



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  		Figure 1. Transient Transfection of InhBP into CV-1 Cells Confers Inhibin Binding Ability

A, The Flag-tagged full-length InhBP cDNA was transfected into CV-1 cells, and the expressed protein was localized to membranes by immunofluorescence analysis using the anti-Flag antibody. B–E, To determine whether InhBP can confer inhibin binding to CV-1 cells, Flag-tagged InhBP-transfected and nontransfected cells were incubated with iodinated inhibin A (D and E, dark-field) or inhibin B (H and I, dark-field) followed by immunolocalization of InhBP-transfected cells with the anti-Flag antibody (B, C, F, and G, bright-field). No inhibin binding was detected in nontransfected cells (B, D, F, and H). Arrows in B and C and F and H denote same cell. Inhibin binding was detected specifically in those cells that expressed InhBP (C, E, G, and I).

 
Assembly of InhBP into Homooligomeric Complexes
Several groups have reported that both type I and type II TGFß serine-threonine kinase receptors can assemble into homooligomeric complexes in vivo and in vitro (29, 30, 31). Therefore, the ability of InhBP to form homooligomers was determined in transiently transfected HeLa cells. In addition, the role of inhibin and activin in regulating assembly of higher order complexes of InhBP was examined. InhBP was capable of forming ligand-independent homooligomers (Fig. 2AGo). The formation of homooligomeric complexes was unaffected by treatment of the cells with inhibin A, inhibin B, or activin A alone, and addition of inhibin A and activin A together did not alter the complex. However, InhBP homooligomer assembly was partially disrupted upon treatment with a combination of inhibin B and activin A.



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  		Figure 2. InhBP Assembles into Complexes with Activin Receptor Subunits

A, InhBP forms homooligomeric complexes in a ligand-independent manner (upper panel). HeLa cells were transiently transfected with equal amounts of Flag-tagged InhBP and HA-tagged InhBP and treated with 100 ng/ml of inhibin A, inhibin B, or activin A as indicated 30 min before lysis, and lysates were subjected to immunoprecipitation with anti-HA antibody and immunoprecipitated complexes were analyzed by SDS-PAGE, immunoblotting with anti-Flag antibody and detected by chemiluminescence. Protein expression was confirmed by immunoblotting total cell lysates with anti-HA or anti-Flag antibodies (lower panels). Closed arrows indicate protein relative molecular mass markers; open arrows indicate migration of proteins. B, InhBP associates strongly with Alk4 and to a lesser extent with Alk2, ActRII, and ActRIIB (upper panel). HeLa cells were transiently transfected with equal amounts of Flag-tagged InhBP and HA-tagged type I (Alk2), type IB (Alk4), type II, or type IIB activin receptors. Cells were incubated with 100 ng/ml inhibin A for 30 min before lysis. Lysates were subjected to immunoprecipitation, immunoblotting, and analysis as in panel A. Protein expression was confirmed by immunoblotting total cell lysates with anti-HA or anti-Flag antibodies (lower panels). Closed arrows indicate protein relative molecular mass markers; open arrows indicate migration of proteins. C, Truncation of the C-terminal serine-threonine kinase domain of Alk4 does not affect assembly with InhBP (upper panels). HeLa cells were transiently transfected with equal amounts of Flag-tagged InhBP and myc-tagged Alk4 C-terminal domain truncation mutant (Alk4{Delta}CT). Lysates were subjected to immunoprecipitation with anti-myc antibody and immunoprecipitated complexes were analyzed as in panel A. Protein expression was confirmed by immunoblotting total cell lysates with anti-myc or anti-Flag antibodies (lower panels). Closed arrows indicate protein relative molecular mass markers; open arrows indicate migration of proteins. D, Endogenous assembly of InhBP and Alk4 in human pituitary extracts. Three samples of human pituitary tissue were prepared, and 2 mg of total lysate were subjected to immunoprecipitation with anti-InhBP antibody as indicated. Immunoprecipitated complexes were analyzed by SDS-PAGE on a 4–12% bis-acrylamide Tris gradient gel (Novex, San Diego, CA), immunoblotted with anti-ActRIB antibody (R&D Biosystems), and detected by chemiluminescence (upper panels). Protein expression was confirmed by immunoblotting total cell lysates with anti-InhBP or anti-ActRIB antibodies (lower panels). HeLa cells were transiently transfected with Alk4 or InhBP, and protein expression was confirmed by immunoblotting total cell lysate concurrently with the human pituitary samples. HeLa cells express low levels of Alk4 and no detectable InhBP, and complexes of endogenous HeLa Alk4 and expressed InhBP were not observed (upper panel, lane 2). Closed arrows indicate protein relative molecular mass markers; open arrows indicate migration of proteins. Asterisk denotes nonspecific protein-anti-Alk4 antibody interaction that runs just below InhBP/Alk4 band (arrowhead, upper panel).

 
Assembly of InhBP and Activin Receptor Subunit Complexes
Based on the fact that InhBP appears to have no kinase domain and may rely on extrinsic serine-threonine kinase receptors to transduce a signal, and because inhibin is known to bind with low affinity to the activin type II receptor, functional assembly of InhBP with known TGFß family member receptor subunits was examined. Flag-tagged InhBP and various hemagglutinin (HA)-tagged or myc-tagged activin receptors were coexpressed in HeLa cells, and the cells were treated with 100 ng/ml inhibin A for 30 min before lysis. InhBP-activin receptor complexes were coimmunoprecipitated using antibodies directed against the HA or myc epitope tags. A weak interaction between InhBP and activin type II (ActRII) and type IIB (ActRIIB) receptors was detected by immunoblotting with anti-Flag antibody (Fig. 2BGo). A direct association between InhBP and two signaling receptor subunits, Alk4 (activin type IB receptor) and Alk2 (activin type I receptor), was readily observed by immunoprecipitation followed by Western detection (Fig. 2BGo). No interaction between InhBP and HA-tagged GH releasing hormone receptor was detected (data not shown), indicating that the interaction of InhBP with activin receptors is specific. To investigate whether InhBP interacts with the conserved serine-threonine kinase domain of Alk4, a myc-tagged Alk4 expression construct, which lacks the kinase domain, was cotransfected with InhBP (32). A stable InhBP-Alk4 C-terminal deletion mutant (Alk4{Delta}CT) complex was detected by immunoblot, suggesting that the interaction between these two cell surface proteins is dependent on regions lying outside the C-terminal serine-threonine kinase domain of Alk4 (Fig. 2CGo).

In Vivo Association of InhBP and Activin Receptor Type IB
To verify that InhBP can assemble with Alk4 in a physiological context, we immunoprecipitated InhBP- containing protein complexes from three samples of human pituitary lysate using the anti-InhBP antibody. Interaction of endogenous InhBP with Alk4 was observed by immunoblotting with a human anti-Alk4 antibody (Fig. 2DGo, upper panel). Identity of the complexed pituitary proteins was established by comparison of proteins identified in human pituitary to immunoblots of overexpressed InhBP and Alk4 in HeLa cell lysates run concurrently with the pituitary samples (Fig. 2DGo, lower panels). Similarly, InhBP and Alk4 complexes were identified in ovine pituitary lysates (data not shown).

Dynamic Regulation of InhBP and Activin Receptor Subunit Complexes
Because activin A and inhibin B together interrupt the InhBP homooligomer (Fig. 2AGo), the role of these ligands on Alk4 and InhBP complex assembly was investigated. Cells were transfected with Flag-tagged InhBP and HA-tagged Alk4 and treated with 100 ng/ml inhibin A, inhibin B, or activin A for 30 min before lysis (Fig. 3AGo). InhBP and Alk4 can assemble in a ligand-independent manner and activin A does not alter this stable association (Fig. 3BGo). A subtle but consistent decrease in complex assembly was observed when cells were treated with inhibin B alone.



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  		Figure 3. InhBP Associates with the Activin [Type IB] Receptor, Alk4, in a Ligand- Dependent Manner

A, The assembly of InhBP/Alk4 complexes is antagonized in the presence of activin and inhibin B. HeLa cells were transiently transfected with equal amounts of Flag-tagged InhBP and HA-tagged Alk4. Cells were incubated with 100 ng/ml activin A, inhibin A, or inhibin B as indicated for 30 min before lysis. Lysates were subjected to immunoprecipitation with anti-HA antibody as indicated. Immunoprecipitated complexes were analyzed by SDS-PAGE on an 8% acrylamide gel (Bio-Rad Laboratories, Inc.), immunoblotted with anti-Flag antibody, and detected by chemiluminescence. Protein expression was confirmed by immunoblotting total cell lysates with anti-HA or anti-Flag antibodies (lower panels). Closed arrows indicate protein relative molecular mass markers; open arrows indicate migration of proteins. B, Data from densitometric analysis of Fig. 3BGo are presented as percent of ligand-independent (basal) InhBP/Alk4 complex assembly corrected for protein expression levels in the lysate and reported in arbitrary units.

 
To investigate whether the InhBP-Alk4 interaction was altered by coincubation of the antagonistic ligands, cells were treated simultaneously with 100 ng/ml inhibin A or B plus activin A, and complex formation was evaluated (Fig. 3AGo). Association of InhBP and Alk4 was not altered by treatment with inhibin A and activin A together (Fig. 3BGo). However, when cells were treated with inhibin B and activin A, the interaction between InhBP and Alk4 diminished to approximately 60% of the basal associated complex (Fig. 3BGo). These results suggest that a heteromeric cell surface receptor complex is formed that includes InhBP and Alk4, and that formation of this complex is regulated in a dynamic manner by activin and inhibin B.

Modulation of Activin-Regulated Transcriptional Responses by InhBP and Inhibin
Because inhibins antagonize activin action in various physiological settings, the role of InhBP in regulating activin-dependent signal transduction events was investigated. The TSA kidney epithelial cell line used in this study expresses type IB (Alk4) and both type II activin receptors, but does not express InhBP (data not shown), and therefore provides a model system in which to study InhBP action and interaction with an endogenous activin signaling pathway. Furthermore, as no inhibin-specific promoter has yet been identified, a known activin/TGFß responsive reporter gene construct, the plasminogen activator inhibitor-1 promoter ligated to a luciferase reporter (p3TP-luc), was used to investigate the effects of InhBP and inhibin on activin-stimulated gene transcription.

TSA cells were transiently transfected with p3TP-luc and the cells were treated with the indicated concentrations of inhibin, activin, or with inhibin plus activin for 24 h (Fig. 4Go). In our TSA system, activin A was able to stimulate luciferase activity 8- to 10-fold over basal. Treatment of cells with equimolar concentrations of inhibin A and activin A resulted in a 70% antagonism of activin-stimulated transcription of the reporter construct (Fig. 4AGo), while inhibin B was capable of abrogating the activin A effect by approximately 40% (Fig. 4BGo). This antagonism may be attributed to competition between activin and inhibin for binding to the endogenous type II activin receptor in TSA cells (17). Interestingly, transfection of InhBP into this system resulted in ligand-independent antagonism of activin-stimulated p3TP-luc transcription, and the mechanism underlying this effect is currently under investigation. Remarkably, in the presence of InhBP, inhibin B treatment resulted in a nearly total loss (90%) of activin A-stimulated p3TP-luc transcription (Fig. 4BGo), while inhibin A treatment of InhBP-transfected cells had no additional antagonistic effect on the activin response (Fig. 4AGo). The antagonistic effect of inhibin B and InhBP on activin-stimulated p3TP-luc was approximately 2 times greater than that observed with inhibin B treatment alone, and so could not be due to competition for activin receptor binding alone. This result indicates a direct role for InhBP in a novel receptor-based mechanism of antagonism of activin-stimulated p3TP-luc transcription by inhibin B. These results also strongly support the view that there is a fundamental difference in the molecular actions of inhibin A and inhibin B.



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  		Figure 4. InhBP Mediates Inhibin B-Mediated Antagonism of Activin-Stimulated p3TP-luc Transcription in TSA Cells

A and B, TSA cells were transiently transfected with p3TP-luc and either 50 ng pcDNA3.0 or InhBP expression plasmid. Cells were treated with 100 ng/ml inhibin A, inhibin B, or activin A as indicated. Expression of p3TP-luc is calculated as relative light units corrected for total protein in the lysate and is plotted as the percent stimulation of luciferase expression ± SE (s.e.m.) of triplicates from a representative experiment. C and D, Alk4 overexpression opposes InhBP and inhibin B-mediated activin antagonism. Cells were transiently transfected with p3TP-luc and either pcDNA3.0, 50 ng InhBP expression plasmid, or 25 ng of expression plasmid encoding Alk4 as indicated. Cells were treated with 100 ng/ml inhibin A, inhibin B, or activin A as indicated. Expression of p3TP-luc is calculated as relative light units corrected for total protein in the lysate and is plotted as the percent stimulation of luciferase expression ± SE (s.e.m.) of triplicates from a representative experiment. *, P < 0.05; **, P < 0.01. E, Antagonism of p3TP-luc expression by inhibin and InhBP is activin specific. TSA cells were transiently transfected with p3TP-luc and either pcDNA3.0 or 25 ng InhBP expression plasmid and treated with 10 ng/ml TGFß1 or 100 ng/ml inhibin B as indicated. Expression of p3TP-luc is expressed as relative light units normalized for total protein and is plotted as the mean ± SEM of triplicates from a representative experiment.

 
Role of Alk4 in the Modulation of Activin-Stimulated Transcriptional Responses by Inhibin and InhBP
The biochemical data predict a strong, ligand-modulated interaction between InhBP and Alk4. To investigate whether Alk4 is involved in the InhBP/inhibin-mediated antagonism of activin-stimulated p3TP-luc expression, TSA cells were cotransfected with the p3TP-luc reporter construct and an Alk4 expression plasmid, and the cells were treated with inhibin and activin. Overexpression of Alk4 partially reversed the functional antagonism of p3TP-luc expression due to competition between inhibin A and activin A for receptor binding (Fig. 4CGo) and completely reversed antagonism of activin A action by inhibin B (Fig. 4DGo). Notably, Alk4 overexpression only partially restored activin- stimulated p3TP-luc transcription in cells transfected with InhBP and treated with inhibin B, from 90% (Fig. 4BGo) to 56% antagonism (Fig. 4DGo). Thus, overexpression of Alk4 opposes inhibin B and InhBP-mediated activin antagonism in this system.

Modulation of Transcription by Inhibin and InhBP Is Activin Specific
It is imperative to establish the antagonistic effect of inhibin B and InhBP as activin specific. Inhibin A was shown to antagonize activin A-stimulated p3TP-luc expression while having no effect on TGFß-stimulated p3TP-luc transcription in a CHO cell line (19). Importantly and necessarily, the antagonistic effect of InhBP and inhibin B on p3TP-luc transcription is activin specific (Fig. 4EGo). TGFß is capable of stimulating p3TP-luc transcription 8-fold over basal in TSA cells (data not shown). As anticipated, neither inhibin A (data not shown) nor inhibin B alone has a significant antagonistic effect on TGFß-stimulated p3TP-luc transcription (Fig. 4EGo). Unlike the ligand-independent antagonistic effect of InhBP on antagonism of activin-stimulated p3TP-luc transcription, TGFß-stimulated luciferase activity is unaffected by transfection of InhBP into the system. Furthermore, cotransfection of InhBP did not support antagonism of TGFß signal transduction by inhibin B (Fig. 4E).

InhBP Interacts with Other Type I Receptors of the TGFß Superfamily
Cross-talk within the TGFß family occurs frequently through sharing of receptors by multiple ligands and the formation of nontraditional receptor complexes. For example, bone morphogenetic protein 2 (BMP2) and activin are able to signal through Alk2 in some cell types (33), and it has been shown that inhibin can bind, albeit weakly, to the activin type II receptor (17, 19). TGFß and activin bound to their respective type II receptors can form complexes with the type I TSR-1 (Alk1) (34), and BMP7 binds ActRII to form functional complexes with the BMP type I receptors Alk3 and Alk6 (33). Based on these observations, the interaction of InhBP with type I receptor proteins of other TGFß family members, including Alk3 and Alk6, as well as the dual specificity type I receptor Alk1, was investigated. Flag-tagged InhBP and HA-tagged BMP type I receptors or HA-tagged dual type I receptor were coexpressed in HeLa cells (Fig. 5Go). Receptor complexes were coimmunoprecipitated using antibodies directed against the HA epitope tags. A very weak interaction between InhBP and Alk3 was detected by immunoblot using antibody directed against the Flag epitope tag, while InhBP readily formed complexes with Alk1 and Alk6.



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  		Figure 5. InhBP Associates with Other Type I TGFß Superfamily Receptors

InhBP interacts strongly with the dual-specificity type I receptor, Alk1, as well as with the BMP type I receptor, Alk6, and to a lesser extent with the BMP type I receptor, Alk3 (upper panel). HeLa cells were transiently transfected with equal amounts of Flag-tagged InhBP and HA-tagged dual specificity type I receptor (Alk1), or the BMP type I receptors Alk3 or Alk6. Lysates were subjected to immunoprecipitation with anti-HA antibody, and immunoprecipitated complexes were analyzed by SDS-PAGE and immunoblotting with anti-Flag antibody and detected by chemiluminescence. Protein expression was confirmed by immunoblotting total cell lysates with anti-HA or anti-Flag antibodies (lower panels). Closed arrows indicate protein relative molecular mass markers; open arrows indicate migration of proteins.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Gonadal inhibin inhibits pituitary FSH synthesis, promotes ovarian follicle maturation, and is important to the maintenance of normal spermatogenesis. Although our knowledge of how activin acts on target cells has expanded tremendously in the last several years, progress in understanding the mechanism of inhibin action has been comparatively slow. The means by which inhibin regulates distal or local cellular events is gradually being revealed by the identification of cell surface inhibin-binding proteins (27, 28, 35, 36). Biochemical purification of InhBP and the recent characterization of betaglycan as an inhibin-binding protein suggests that inhibin-binding cell surface proteins act as docking molecules that permit assembly of specific receptor complexes which then modulate ligand action (28, 36, 37, 38).

The Ig domain-rich nature of InhBP predicted a mechanism of inhibin signal transduction differing significantly from other members of the TGFß superfamily. Interestingly, Ig domains are found in a wide variety of proteins, including growth factor receptors for epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and hepatocyte growth factor (HGF), and in cell adhesion molecules including neural cell adhesion molecule (N-CAM) and cadherin (39). Furthermore, many members of the Ig superfamily are signal transducers; for example, the Ig-domain containing PDGF receptor (PDGF-R) tyrosine kinase initiates intracellular kinase cascades that lead to cell division and proliferation (reviewed in Ref. 40), while activation of N-CAM has been shown to result in changes in intracellular second messenger levels and protein phosphorylation (reviewed in Ref. 41).

Further, the lack of an intracellular kinase domain suggested that InhBP would require association with other proteins or extrinsic kinases to transduce an inhibin signal. We observed that InhBP is capable of interacting with the activin type IB receptor serine-threonine kinase in a ligand-dependent manner. This observation was unexpected because it was hypothesized that inhibin binds InhBP and ActRII cooperatively through its {alpha}- and ß-subunits, respectively. Accordingly, Lewis et al. (36) have reported that betaglycan binds inhibin and disrupts activin signal transduction through association with the activin type II receptor (Fig. 6BGo). Similarly, interaction between ligand-bound type II TGFß receptor and the accessory signaling molecule endoglin interferes with TGFß signal transduction (Fig. 6CGo) (42).



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  		Figure 6. InhBP and Inhibin B Antagonize Activin Signal Transduction by Modulating Activin Receptor Complex Assembly

A, InhBP is assembled into the activin heteromeric receptor complex through its ligand-independent association with Alk4, thus rendering the complex inhibin B sensitive. Inhibin B binding to InhBP disrupts the association of InhBP, Alk4, and ActRIIB and abolishes activin signaling. B, Betaglycan binds inhibin A in cooperation with ActRII, thereby disrupting heteromeric activin receptor complex formation and activin signaling. C, Endoglin binds TGFß in the presence of the TGFß type II receptor (TßRII), which disrupts complex formation with TGFß type I receptor (TßRI) and interferes with TGFß signal transduction.

 
However, it is the interaction between InhBP and an activin type I receptor, Alk4, that is central to inhibin B action. InhBP associates strongly with Alk4 in vivo and in vitro in the absence of ligand, and this complex is stabilized in the presence of activin. To our knowledge, interaction of Alk4 with InhBP is the first example of in vivo assembly of any of the TGFß superfamily receptors. Through this interaction with Alk4, we hypothesize that InhBP is recruited to the Alk4/ActRII/B activin receptor complex. Addition of inhibin to the system destabilizes the association between InhBP and Alk4 and abrogates activin signal transduction through its receptor complex (Fig. 6AGo).

One of the unique features of inhibin is that it specifically antagonizes some actions of activin but not those of other TGFß superfamily ligands. One mechanism by which inhibin opposes activin action is through binding interference of the inhibin ß-subunit to the activin type II receptor (17). In our study, functional antagonism of activin-stimulated p3TP-luc was observed when cells were treated with equimolar amounts of activin A and inhibin A or inhibin B, which resulted in approximately 50% inhibition of p3TP-luc expression. LeBrun and Vale (18) have shown that inhibin abrogates activin-stimulated p3TP-luc expression in K562 cells, but that in a cell line which inducibly overexpresses the activin type IB and type II receptors (KAR6), inhibin can no longer antagonize activin action even at 8-fold molar excess. Thus, it was proposed that an additional inhibin receptor or other accessory molecule was necessary to fully antagonize activin action. Our results support this hypothesis. Cells treated with inhibin A or inhibin B alone resulted in a partial antagonism of activin-stimulated p3TP-luc, while inhibin B treatment in the presence of cotransfected InhBP virtually abolished activin-stimulated p3TP-luc expression. Molecular and functional models of inhibin antagonism require that the concentration of inhibin {alpha}-subunit or inhibin dimer is such that it blocks the activin signal transduction apparatus through abrogation of activin dimer assembly and binding to its receptor. The data suggest that InhBP provides a means through which low levels of inhibin B can antagonize activin signal transduction, whereby the presence of InhBP in activin-stabilized receptor complexes renders these complexes sensitive to antagonism by inhibin B (Fig. 6AGo).

Accordingly, overexpression of Alk4 in the system reverses activin-stimulated p3TP-luc expression, perhaps by shifting the stoichiometry of InhBP and Alk4 and favoring the assembly of activin receptor complexes that do not contain InhBP. These complexes would be inhibin B insensitive and remain intact upon inhibin B treatment. The inability of excess inhibin to antagonize activin signaling in models such as the activin receptor- overexpressing KAR6 cell line may be due to a lack of endogenous InhBP in these cells or the assembly of a greater number of activin receptor complexes that do not contain InhBP.

It is apparent that InhBP may be an inhibin B receptor. Although InhBP is capable of binding to both inhibin A and inhibin B, antagonism of activin-stimulated p3TP-luc transcription by inhibin A was not enhanced by the coexpression of InhBP, while inhibin B and InhBP were able to abrogate activin signaling more than 90%. Once thought to be interchangeable, inhibin A and inhibin B have very different synthesis and secretion patterns during the female reproductive cycle (8, 43, 44). Furthermore, inhibin B, rather than inhibin A, predicts follicle health and is an early indicator of ovarian aging (45, 46). In addition, inhibin A and inhibin B are produced in a sexually dimorphic manner: inhibin B, but not inhibin A, is produced by the testes and is inversely correlated to FSH in several experimental models as well as in disease states (8, 47, 48). Thus, the two species of inhibin exist in discrete molecular, cellular, and endocrine niches. The observation that InhBP mediates antagonism by inhibin B and not inhibin A suggests that there is also a fundamental, functional difference between the two species of inhibin. The basis for this difference remains unclear, but may be partially attributed to the structural dissimilarity of the inhibin ß-subunits, ßA and ßB, which share 63% identity. Likewise, TGFß1 and TGFß2 exhibit approximately 80% amino acid identity, yet they are regarded as functionally distinct proteins. Comparison of these isotypes of TGFß in several studies revealed that TGFß1 has a higher binding affinity for the type II TGFß receptor and is a more potent inhibitor of hematopoietic progenitor cell and endothelial cell proliferation than TGFß2 (49, 50, 51).

Furthermore, it is important that antagonism of p3TP-luc transcription by InhBP and inhibin B is activin specific. The effects seen on p3TP-luc expression upon treatment with inhibin B and activin are not repeated when cells are treated with TGFß. Although InhBP interacted with the TGFß dual specificity type I receptor Alk1, inhibin B and InhBP had no effect on TGFß-stimulated p3TP-luc transcription. Thus, antagonism of activin action by InhBP is a dynamic, ligand-dependent process in which InhBP provides more than just a physical blockade to signaling through the activin receptor.

These results, along with the fact that inhibin A and inhibin B are differentially regulated throughout the human menstrual and rat estrous cycles, support the existence of separate inhibin A and inhibin B receptors within the female reproductive axis (8, 43, 52). In rat, ovarian inhibin A subunit mRNA and protein levels remain low through metestrus and diestrus, then rise to peak late in proestrus at the time of the primary gonadotropin surges (8, 52). Conversely, a high concentration of inhibin B is maintained through metestrus and diestrus, then falls to its lowest levels after the primary FSH surge and remains low through the secondary FSH surge (8). Activin A levels throughout the cycle remain virtually undetectable until peaking sharply just before the primary and secondary FSH surges. High levels of inhibin B early in the cycle keep FSH low until the drop in inhibin B levels coupled with GnRH and an activin peak in proestrus permit the FSH surge and ovulation. Preliminary data from our laboratory suggest that in the rat, pituitary InhBP expression closely follows inhibin B expression, lending further support to the characterization of InhBP as an inhibin B receptor (52A ).

Betaglycan was recently identified as an inhibin A-binding protein (36). Several features of this cell surface molecule differ from InhBP (53), indicating that different mechanisms of inhibin action may be available to a variety of cell types. Unlike InhBP, which disrupts receptor complex formation in the presence of inhibin B, betaglycan binds and sequesters type II activin receptors in the presence of inhibin A (Fig. 6BGo). Cells that express both betaglycan and InhBP are predicted to be exquisitely sensitive to the actions of inhibin. A potential example of this type of cell is the pituitary gonadotrope. Indeed, in humans, the gonadotrope may be able to respond to both early follicular inhibin B via InhBP and luteal phase inhibin A via betaglycan. Of course, this hypothesis will need to be tested in vivo. Lastly, the testis secretes inhibin B and not inhibin A. Thus, we predict that InhBP will be the dominant inhibin receptor in the male pituitary.

Finally, it is interesting that both InhBP and endoglin share the ability to assemble with various type I receptors of the TGFß superfamily. However, in general, endoglin interacts with ligand binding receptors within the superfamily (54), while InhBP can interact with both activin and TGFß signaling type I receptors, such as Alk4 and Alk1, and with the BMP2 ligand binding type I receptor, Alk6. Furthermore, while endoglin can only interact with various ligand binding receptors in the presence of ligand, the role of ligand in assembly of InhBP with type I receptors remains unclear.

In conclusion, the recently purified and cloned high-affinity inhibin-binding protein, InhBP, is a crucial component of a heteromeric receptor complex that can specifically modulate activin-stimulated reporter gene expression in a model cell line. Despite its unique properties, InhBP appears to be intricately involved in inhibin signal transduction events through modulation of traditional activin receptor complexes. Antagonism of activin action occurs through receptor complex assembly-based mechanism whereby ligand-bound InhBP disrupts formation of active activin receptor complexes. These data do not rule out other means of activin antagonism mediated by InhBP and inhibin. Future endeavors will focus on the identification of other protein components of the InhBP signal transduction complex, including the Smad proteins, as well as the identification of inhibin-responsive genes. Taken together, these studies will help us to better understand inhibin and InhBP signal transduction and its role in normal reproductive function and gonadal oncogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Recombinant Ligands
Recombinant human (rh) inhibin A, inhibin B, activin A, and TGFß1 were purchased from R&D Biosystems (Minneapolis, MN), Genentech, Inc. (South San Francisco, CA), or were produced in the laboratory. The ligands were formulated in a buffer of 0.15 M NaCl and 0.05 M Tris, pH 7.5.

Immunofluorescence and Immunohistochemistry
The monkey kidney CV-1 cell line was transiently transfected with the Flag-tagged InhBP and subjected to immunofluorescence analysis using the mouse anti-Flag antibody (Sigma, St. Louis, MO) followed by a rabbit-antimouse fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Pierce Chemical Co., Rockford, IL). Immunohistochemical studies were performed using the rabbit anti-InhBP antisera or mouse anti-Flag antibody (Sigma), followed by a biotinylated goat antimouse secondary antibody or goat antirabbit secondary antibody (Vector Laboratories, Inc., Burlingame, CA), washed, and incubated with ABC reagent (Vectastain ABC kit, Vector Laboratories, Inc.). Signal was detected using the DAB substrate kit (Vector Laboratories, Inc.).

In Situ Ligand Binding
Inhibin was labeled using a modified lactoperoxidase method. Briefly, 5 µg of ligand were diluted in 0.4 M sodium acetate, pH 5.6, and 0.5 nmol Na125I (0.5 nmol/mCi on calibration date), 0.5 IU lactoperoxidase, and 0.25 nmol H2O2 were added sequentially. The ligand was incubated at ambient temperature with intermittent vortexing for 5 min. The reaction was quenched with 450 µl PBS + 0.05% Tween 20 + 0.5% BSA (Intergen, Purchase, NY). A 10 µl aliquot of the precolumn fraction was removed for trichloroacetic acid (TCA) precipitation. Free iodine was removed using Sephadex G-10 column chromatography (PD-10, Pharmacia Biotech, Piscataway NJ). The specific activity of the ligands used in the binding studies was approximately 100 µCi/µg. The biological activity of the iodinated ligand was determined using male rat anterior pituitary cells. Iodinated inhibin (40 pM) was added to cells for 12 h at room temperature. Binding to InhBP-transfected CV-1 cells was detected by exposure of emulsion and analyzed by dark-field microscopy.

Immunoprecipitation and Immunoblot Analysis
Cells were transfected with various receptor constructs using the Vaccinia-T7 RNA polymerase hybrid expression system. HeLa cells were infected with Vaccinia virus vTF7.3 expressing the bacteriophage T7 RNA polymerase (obtained under license from Dr. Bernard Moss, NIH, Bethesda, MD), for 1 h and the various plasmid DNA/liposome mixtures were added and incubated for 14 h. Cells were treated with ligand for 0.5 h before lysis. Cells were lysed in RIPA buffer [50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton-X-100, 0.5% deoxycholic acid, and 0.1% SDS plus protease inhibitors (Roche Molecular Biochemicals, Mannheim, Germany)]. The lysates were clarified by centrifugation and incubated with mouse HA-specific antibody 12CA5 (kindly provided by Dr. Robert A. Lamb, Northwestern University) overnight at 4 C. Immunocomplexes were precipitated by protein A-agarose beads (Vector Laboratories, Inc.) for 1.5 h and separated in a 8.5% SDS-PAGE gel (Bio-Rad Laboratories, Inc. Hercules, CA) or in a 4–12% gradient bis-acrylamide-Tris NuPAGE gel (Invitrogen/Novex, Carlsbad, CA). Proteins were transferred to nitrocellulose membrane (Bio-Rad Laboratories, Inc.), blotted with mouse anti-Flag M2 antibody (Sigma), mouse anti-HA antibody, mouse anti-c-myc antibody (Sigma), or goat anti-hActRIB (Alk4) antibody (R&D Biosystems), for 1 h at room temperature, followed by 1 h incubation in horseradish peroxidase (HRP)-conjugated goat antimouse antibody (Bio-Rad Laboratories, Inc.), HRP-conjugated donkey antirabbit antibody (Amersham Pharmacia Biotech, Buckinghamshire, UK), or HRP-conjugated antigoat antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at room temperature and detection by chemiluminescence (Amersham Pharmacia Biotech).

DNA Constructs
The p3TP-luc reporter plasmid was provided by the J. Massague laboratory. Activin receptor (type I, IB, II, and IIB) expression plasmids were provided by the L. Mathews and K. Mayo laboratories and modified by insertion of HA or Flag sequence at the 3'-end. HA-tagged Alk1, Alk3, and Alk6 were a generous gift from the L. Attisano laboratory and were modified by subcloning into the pcDNA3 vector (Invitrogen). The myc-tagged Alk4 C-terminal deletion mutant was donated by the W. Vale laboratory.

Transient Transfection and Luciferase Assays
TSA cells were maintained in DMEM high glucose media (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FCS (Life Technologies, Inc.) and 1% antibiotic/antimycotic (Life Technologies, Inc.) and incubated at 37 C, 5% CO2. Cells were plated 1 day before transfection in 24-well plates at 1.2 x 105 cells per well and transiently transfected with p3TP-luc and various expression plasmids encoding various activin, TGFß, and BMP receptors or empty pcDNA3 vector (Invitrogen) using the calcium phosphate transfection method (55). After an overnight recovery, cells were treated with indicated ligands for 24 h in DMEM high-glucose media, 1% antibiotic/antimycotic. Cells were lysed in Triton-X-100, and luciferase activity was measured using standard techniques. Total protein was measured using BCA reagent (Bio-Rad Laboratories, Inc.).


   ACKNOWLEDGMENTS
 
We would like to thank Wei Chen, Huira Chong, and Kelly Mayo for work on the initial cloning of InhBP. Many thanks to the Attisano/Wrana Laboratory (University of Toronto, Toronto, Canada), the Vale Laboratory (The Salk Institute for Biological Studies, La Jolla, CA), the Mathews Laboratory (University of Michigan, Ann Arbor, MI), the Massague Laboratory (Memorial Sloan-Kettering Cancer Center, New York, NY), and the Mayo Laboratory (Northwestern University, Evanston, IL) for their generous gifts of the various receptor constructs, as well as Robert A. Lamb (Northwestern University) for kindly providing the mouse anti-HA antibody. We would also like to thank Patrick Sluss (Massachusetts General Hospital, Boston, MA) for human pituitary tissue. Thanks to Huira Chong for cloning of plasmid constructs, InhBP immunofluorescence, and in situ ligand binding studies. We would also like to thank Brad Draper for preparation of plasmid constructs, Stephanie Pangas for purified ligands, and Daniel J. Bernard and Fernando Lopez-Casillas for helpful discussions.

This study was supported by NIH Grants HD-37096 and HD-28048 to T.K.W. S.C.C. is a fellow of the Northwestern University Cellular and Molecular Basis of Disease Training Grant (GM-08061).


   FOOTNOTES
 
Address requests for reprints to: Teresa K. Woodruff, Department of Neurobiology and Physiology, Northwestern University, O.T. Hogan 4–150, 2153 North Campus Drive, Evanston, Illinois 60208. E-mail: tkw{at}northwestern.edu

Received for publication October 6, 2000. Accepted for publication December 13, 2000.


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