Antagonists of activin signaling: mechanisms and potential biological applications
Craig A. Harrison1,2, Peter C. Gray2, Wylie W. Vale2 and David M. Robertson1
1Prince Henry’s Institute of Medical Research, 246 Clayton Road, Clayton, VIC 3168, Australia
2Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA

Activins are members of the transforming growth factor-b (TGF-b) superfamily that control many physio- logical processes such as cell proliferation and differen- tiation, immune responses, wound repair and various endocrine activities. Activins elicit these diverse biologi- cal responses by signaling via type I and type II receptor serine kinases. Recent studies have revealed details of the roles of inhibin, betaglycan, follistatin and its related protein follistatin-related gene (FLRG), Cripto and BAMBI in antagonizing activin action, and exogenous antagonists against the activin type I (SB-431542 and SB-505124) and type II (activin-M108A) receptors have been developed. Understanding how activin signaling is controlled extracellularly is the first step in providing treatment for wound healing and for disorders such as cachexia and cancer, which result from a deregulated activin pathway.

Introduction
The structurally related proteins of the TGF-b family control diverse cellular processes including cell prolifer- ation, cell death, metabolism, homeostasis, differen- tiation, immune responses and endocrine function [1–4]. The disruption or deregulated activity of TGF-b super- family members is often associated with pathological states such as aberrant cellular differentiation, prolifer- ation and metabolism [5,6]. Activins are typical members of the TGF-b superfamily in that they possess a cysteine knot scaffold and are secreted as homo- or heterodimers of related b-subunits. Although four b-subunit genes (bA, bB, bC and bE) have been described in human, only dimers of bA and bA (activin A), bB and bB (activin B), and bA and bB (activin AB) have been shown to be biologically active. Activins signal by interacting with two transmembrane serine/threonine receptor kinases (type I and type II receptors; Table 1). Activin binds initially to its type II receptor, ActRII (for activin A) or ActRIIB (for activin B), which leads to the recruitment, phosphorylation and subsequent activation of the type I receptor, activin-like kinase 4 (ALK4). On activation, ALK4 binds and then phosphorylates a subset of cytoplasmic Smad proteins (Smad2 and Smad3; Table 1), which form part of the

Corresponding author: Harrison, C.A. ([email protected]). Available online 27 January 2005
post-receptor signal transduction system [7]. Access of activin to the cell is regulated by various extracellular binding proteins.
In this review, we focus on the recently identified mechanisms of action of the known endogenous activin antagonists. We also discuss recent advances in the development of exogenous activin receptor antagonists and their potential biological applications.

Activin antagonists
Inhibin and betaglycan
The activin bA and bB subunits can heterodimerize with the inhibin a-subunit to form inhibin A (a-bA) and inhibin B (a-bB). It is well established that inhibins are crucial for maintaining normal function in many tissues, particularly those of the reproductive axis. In these tissues, inhibin opposes most, but not all, of the actions of activin. As testimony to the importance of inhibin as an antagonist of activin, mice deficient in the inhibin a-subunit develop gonadal tumors and cachexia with severe weight loss and liver necrosis [8,9].
Inhibin antagonism of activin signaling is dependent on competition for binding to type II activin receptors [10,11]. Gray et al. [12] have shown that activin and inhibin share the same binding site on ActRII, supporting the idea that both proteins bind ActRII via their respective b-subunits. The affinity of inhibin for ActRII is, however, about tenfold lower than that of activin [10], and in some tissues and cells inhibin does not antagonize activin, suggesting that additional components are required for inhibin action [13]. Lewis et al. [13] recently identified betaglycan (a TGF-b type III receptor) as a co-receptor of inhibin. Betaglycan binds inhibin with high affinity, and this binding affinity increases about 30-fold in cells coexpressing ActRII and betaglycan. Affinity labeling experiments show that inhibin forms complexes with both recombinant and endogenously expressed betaglycan and ActRII, and that ALK4 is excluded from this ternary complex (Figure 1). Notably, expression of betaglycan can confer inhibin responsiveness in AtT20 corticotrope cells, which are
normally refractory to this hormone [13].
Recent studies have shown that betaglycan protein and mRNA are expressed in rat brain, pituitary and gonads, supporting the idea that betaglycan has a modulatory role

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Table 1. Signaling pathway of activin and selected members of the TGF-b superfamily
Ligand Type II receptor Type I receptor Co-receptor Pathway Smad Common Smad Inhibitory Smad
Activin A ActRII ALK4 (ACTRIB) Smad2 Smad4 Smad7
Activin B ActRIIB ALK7 Smad3
Activin AB
Inhibin A ActRII Betaglycan
Inhibin B ActRIIB
BMPRII
BMP-2 ActRII ALK2 (ActRI) Smad1 Smad4 Smad6
BMP-4 ActRIIB ALK3 (BMPRIA) Smad5 Smad7
BMP-7 BMPRII ALK6 (BMPRIB) Smad8
TGF-b1 TGFbRII ALK5 (TGFbRI) Betaglycan Smad2 Smad4 Smad7
TGF-b2 ALK1 (TSR1) Smad3
TGF-b3
Myostatin ActRII ALK4 (ACTRIB) Smad2 Smad4
ActRIIB ALK5 (TGFbRI) Smad3
Nodal ActRII ALK4 (ACTRIB) Cripto Smad2 Smad4
ActRIIB ALK7 Smad3
Abbreviation: TSR1, TGF-b superfamily receptor type 1.

on inhibin effects in these tissues [14,15]. Because betaglycan-null mice die from lethal proliferative defects in the heart and apoptosis in the liver soon after embryonic day 13.5, however, it has been difficult to confirm that betaglycan has an important role in inhibin action in vivo [16]. The field awaits the development of conditional knockout mice that could be used to focus on blocking betaglycan expression in pituitary gonadotropes or selected gonadal cell types.

Follistatin
Follistatin binds activin with high affinity (50–500 pM) to form biologically inactive complexes, thereby regulating processes as diverse as cell growth and the secretion of follicle-stimulating hormone (FSH) [17,18]. Follistatin consists of a 63-residue amino (N)-terminal segment, followed by three successive follistatin domains of 73–75
amino acids containing ten cysteine residues termed FS-1, FS-2 and FS-3. Alternative splicing generates two follis- tatin isoforms: FS288, which binds heparan sulfate proteoglycans with high affinity via residues in FS-1
[19] and is considered to be a local regulator of activin action; and FS315, which does not bind cell-surface proteoglycans and is the predominant circulating form of the protein.
In addition to its involvement in binding to the cell surface, FS-1 is also essential for suppressing activin biological activity. Keutmann et al. [20] have shown that deletion of FS-1 or the adjacent FS-2 abolishes activin binding, whereas deletion of FS-3 is tolerated. This finding complements earlier studies [21] that established that essential determinants for activin binding are located in the 63-residue N-terminal region that precedes the follistatin domains.

Figure 1. Model of antagonism of signaling by activin and related ligands. Activin, myostatin, Nodal and BMPs signal via ActRII or ActRIIB. The access of these TGF-b superfamily members to their type II receptors is blocked by extracellular binding proteins (follistatin and FLRG), membrane-bound pseudoreceptors (BAMBI and Cripto; for activin only, Cripto actually facilitates Nodal signaling) and structurally related molecules (inhibin and betaglycan). Recent studies using an antagonist of the ActRII and ActRIIB receptor (activin Met108Ala), small-molecule inhibitors of ALK4 (SB-431542 and SB-505124), neutralizing antibodies and the soluble ActRII ECD suggest that it will be possible to achieve pharmacological blockade of activin.

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Surprisingly less is known about the regions of activin involved in follistatin binding. Fischer et al. [22] have recently shown that an activin A deletion mutant, termed des(85–109)-activin A, shows low affinity for both recep- tors and follistatin, suggesting that this region either is directly involved in follistatin binding or lies close to the binding interface. Binding of follistatin to this region would mask residues that are important in the interaction of activin with both ActRII (Ser90, Leu92 and Lys102) and ALK4 (Met91, Ile105 and Met108) [23–25] (Figure 2).
Notably, follistatin seems to have an affinity for activin B that is about ten times lower than its affinity for activin A, suggesting that the determinants for follistatin binding differ between the two isoforms of activin [26]. Further mutagenesis studies are required to elucidate the activin– follistatin interaction in more detail. The generation of an activin mutant that retains high affinity for its receptor but has compromised follistatin binding would act as a superagonist and clearly would have clinical relevance.

Follistatin-related gene
Follistatin-related gene (FLRG), also known as follistatin- related protein or follistatin-like 3, is a recently charac- terized member of the follistatin family that binds activin with relatively high affinity (w850 pM) [27]. FLRG differs from follistatin in lacking the third follistatin domain and a consensus heparin-binding sequence. In this regard, FLRG is thought to act as a circulating activin-binding protein, much like FS315. Outside the 26 conserved cysteine residues, murine FLRG and follistatin share about 40% amino acid identity. Given that both proteins bind and neutralize activin, it seems likely that the

residues that are important for activin binding are located in these conserved regions.
Intriguingly, expression and regulation studies suggest that follistatin and FLRG might not be complete func- tional homologs. FLRG is highly expressed in placenta, testis, skin and cardiovascular tissue, whereas follistatin expression is considerably higher in pituitary and ovary [19]. Wankell et al. [28] have shown that follistatin and FLRG are both expressed during the wound healing process, but their distribution within the wound differs and distinct factors regulate their expression.

Cripto
Nodal, a member of the TGF-b superfamily that is involved in specification of the anterior–posterior and left–right body axes [29,30], has been shown to signal via ActRII or ActRIIB and ALK4. Unlike activins, however, Nodal requires additional co-receptors from the epidermal growth factor, Cripto, FRL-1 and Cryptic (EGF-CFC) protein family, such as Cripto, to assemble its signaling receptor complex [31,32]. Cripto independently binds Nodal via its EGF-like domain and ALK4 via its CFC domain [33] to promote Nodal signaling.
Whereas expression of Nodal is predominantly restricted to the embryo, Cripto is highly overexpressed in breast, pancreatic, colon and ovarian carcinomas [34], raising the possibility that Cripto might regulate the activity of other TGF-b superfamily members in malig- nant adult tissues [35]. Because activins have been shown to be tumor suppressors for breast, liver and kidney cells [36], two groups have recently examined the effects of Cripto on activin signaling. Gray et al. [37] and Adkins
et al. [35] have demonstrated that activin signaling can be

Figure 2. Diagram of the activin-A dimer, highlighting residues involved in interactions with its receptors and binding proteins. Activin contains a cystine-knot fold and a single disulfide bond that covalently connects the two chains of the dimer. In each monomer, two pairs of anti-parallel b-sheets stretch out from the cystine-knot core to form a short (finger 1) and long finger (finger 2). Residues present at the ActRII or ActRIIB interface (red) cluster in these finger regions. In the activin dimer, residues from both the long a-helix (green) and the concave surface of finger 2 (dark blue) have been proposed to form the ALK4-binding site. The important methionine residue at position 108 and the proposed follistatin-binding domain (residues 85–109) are indicated. The residues involved in the interaction between activin with Cripto are unknown. Figure adapted, with permission, from Ref. [61].

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blocked by overexpression of Cripto in numerous cell lines (Figure 1).
Gray et al. [37] did not detect activin binding to Cripto in the absence of ActRII or ActRIIB, suggesting that
activin, unlike Nodal, requires type II receptors to bind Cripto. Their results support a mechanism in which Cripto binding to the activin–ActRII (or activin–ActRIIB) complex inhibits subsequent recruitment of the signaling receptor ALK4. By contrast, Adkins et al. [35] demon- strated that soluble Cripto and activin B interact directly, but they did not detect an interaction between Cripto and activin A. In their model, Adkins et al. [35] propose that the independent binding of Cripto to ALK4 or activin B, or the formation of a complex of activin B, ALK4 and Cripto, is sufficient to inhibit activin signaling.
Although further experiments are required to decipher the molecular mechanisms of Cripto function, the work of these two groups [35,37] has clearly identified a novel extracellular regulator of activin signaling.

BAMBI
The transmembrane protein BAMBI, named after Xeno- pus bone morphogenetic protein (BMP) and activin membrane-bound inhibitor, has sequence similarity to type I receptors of the TGF-b superfamily (53% similarity to Xenopus ALK3) but lacks the intracellular kinase domain required for signaling [38]. BAMBI inhibits signaling by TGF-b superfamily ligands by stably associ- ating, in a ligand-independent manner, with both type I and type II receptors [38]. It has been suggested that the action of BAMBI might be relevant to limiting the
signaling range of BMPs and activins, which act as
morphogens during early embryogenesis [38].
A homolog of BAMBI, referred to as non-metastatic gene A (nma), has been identified in mouse, rat, human and zebrafish. Inhibition of growth in response to TGF-b is restored in several human gastric carcinoma cell lines after treatment with nma antisense oligonucleotides [39]. This finding suggests that nma might play an important role by providing cancer cells with an escape mechanism from TGF-b-mediated growth control.
Recently, Loveland et al. [40] carried out an extensive analysis of BAMBI expression in juvenile and adult rat tissue, with a particular focus on reproductive organs. They demonstrated that the expression of BAMBI mRNA
is regulated in male germ cells, a finding that correlates well with their previous observation that a decrease in activin activity has a role in gonocyte differentiation [40]. Interestingly, the absence of its mRNA in adult smooth muscle and its very low levels in the liver suggest that BAMBI does not regulate the growth inhibitory actions of myostatin and activin, respectively, in these tissues.

Activin type II receptor antagonist
Above, we have described the endogenous mechanisms whereby the activin signaling cascade can be modulated. There is, however, a growing need for the development and use of exogenous antagonists that can block activin signaling. To this end, we and our co-workers [25] recently used a mutagenesis strategy to identify activin A mutants that could bind ActRII but not ALK4, and that would therefore
act as type II activin receptor antagonists. We found that the most important residues for activin biological activity and binding to ALK4 are residues Met91, Ile105 and Met108 on the concave face of finger 2 (Figure 2). These residues, in combination with Tyr35, form a pocket on the surface of activin A that presumably accommodates one or more of the hydrophobic residues on ALK4 (Leu40, Ile70, Val73, Leu75 and Pro77) that are known to be involved in activin binding [41].
Of the activin mutants generated, the Met108Ala variant showed the lowest signaling activity while retain- ing wild-type-like affinity for ActRII [25]. Unlike activin A, the Met108Ala mutant was unable to form a crosslinked complex with ALK4 in the presence of ActRII, indicating that its ability to bind ALK4 was compromised. These data suggested that the Met108Ala mutant might be capable of modulating the signaling of activin and related ligands that use ActRII or ActRIIB. Indeed, signaling in 293T cells in response to activin and myostatin, but not to TGF-b, was reduced in a dose-dependent manner by the Met108Ala variant, consistent with its proposed action as a selective antagonist of type II activin receptors [25].
Future studies should focus on the in vivo activity of the Met108Ala activin mutant. Would overexpression of Met108Ala in muscle lead to antagonism of myostatin and an increase in muscle mass? In a liver regeneration model, would treatment with Met108Ala block activin- induced growth suppression?

Activin type I receptor antagonist
Small-molecule inhibitors have the potential to be used as therapeutics to block signaling pathways that contribute to human diseases. Thus, there is considerable interest in two recently developed competitive inhibitors of the ATP- binding site of ALK5 (a TGF-b type I receptor) [42,43]. These molecules, called SB-431542 and SB-505124, also inhibit ALK4 and ALK7, which share similarity with ALK5 in their kinase domains (Figure 1). They have no effect on ALK1-, ALK2-, ALK3- or ALK6-induced Smad signaling, however, or on the activities of numerous other protein kinases, suggesting that they have a remarkable level of specificity [42,43].
Since its initial characterization, SB-431542 has been shown to inhibit both the TGF-b-induced proliferation of human osteosarcoma cells [44] and the TGF-b-induced promotion of malignant glioma cell proliferation, angio- genesis and motility [45]. It will be interesting to assess the effect of these small-molecule inhibitors in activin- dependent systems.

Neutralizing antibodies and the soluble ActRII extracellular domain
In addition to the recently characterized type I and type II receptor antagonists, several groups have used the extracellular domain (ECD) of ActRII and neutra- lizing antibodies to block activin actions (Figure 1). The ActRII ECD contains all of the structural determinants necessary for high-affinity ligand binding [46]. Treatment of cultured rat anterior pituitary cells with the ActRII ECD [46] or adenovirus-mediated over- expression of the ActRII ECD [47] attenuates secretion of

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FSH in response to exogenous activin A or endogenous activin B. Similarly, antibodies specific for activin A [48] or activin B [49] block the activin-induced stimulation of FSH secretion from pituitary cells and the induction of erythrocyte differentiation.

Potential biological applications of activin antagonism
Cachexia
Mice deficient in the inhibin a-subunit develop sex-cord stromal tumors at an early age [8]. Tumor development is associated with a cachexia-like wasting syndrome, charac- terized by severe weight loss, hepatocellular necrosis around the central vein, and depletion of the parietal cells in the glandular stomach [9]. In inhibin-deficient mice with tumors, activins are increased tenfold in the serum. Coerver et al. [50] have shown that it is the increase in levels of activin signaling via ActRII that is responsible for the cachexia symptoms in these mice.
Notably, the related ligand myostatin has been also implicated in the induction of cachexia. Myostatin is expressed almost exclusively in cells of the skeletal muscle lineage and signals via activin type II receptors (Table 1). Zimmers et al. [51] have shown that systemic overexpression of myostatin in adult mice induces profound muscle and fat loss analogous to that seen in human cachexia syndromes. These studies suggest that activin and myostatin might be useful pharmacological targets for treating cachexia. Indeed, overexpression of follistatin has been shown to slow both activin- and myostatin-induced weight loss [51,52].

Cancer
As mentioned above, targeted deletion of the inhibin a-subunit in mice results in sex-cord stromal tumors at an early age [8]. It seems that the increase in activin in these mice is at least partially responsible for tumor pro- gression, if not development [50,52]. When the inhibin- deficient mice are crossed with mice overexpressing follistatin, serum activin levels are lower and, although the mice still develop gonadal tumors, they do so at a significantly slower rate [52]. These studies suggest that modulating the levels of activin might have beneficial effects on the progression of gonadal tumors.
The benefits of blocking activin action in malignant tissues, however, are likely to depend on the cell context. Activin actually inhibits growth in many types of cell and acts as a tumor suppressor in the early stages of tumorigenesis. Many tumor cells escape the growth inhibitory effect of activin by acquiring mutations in activin receptors [53] or in Smad signaling molecules [54]. Thus, in most tissues activin inhibits the development of cancer and blocking its action would be detrimental. However, activin induces a proliferative response in fibroblasts, keratinocytes and additional gonadal cell types [55] and, in the absence of antagonists, it could be pro-oncogenic in these tissues.

Wound healing
Cutaneous wound repair is a complex process involving blood clotting, inflammation, new tissue formation and finally tissue remodeling. A series of papers by Werner and colleagues [56–58] has implicated activin in these wound

healing processes. Activin is strongly induced in granulation tissue and in suprabasal keratinocytes of the hyperprolifera- tive epithelium after skin injury [56]. This finding led Munz et al. [57] to generate transgenic mice overexpressing the activin bA chain in the epidermis. After skin injury, histological characterization of the wounds revealed a marked increase in the amount of granulation tissue in the wounds of the transgenic mice. Conversely, overexpression of follistatin in the epidermis led to a significant delay in wound healing and a reduction in the formation of scar tissue [58].
These studies show that the levels of activin present in wound tissue are crucial for the outcome of the repair process. Thus, overexpression of activin might be useful for promoting wound healing, whereas inhibition of activin action by receptor antagonists or follistatin could prevent the excessive matrix deposition observed in human skin diseases characterized by either fibrosis or epithelial hyperproliferation.

Concluding remarks
There is currently great interest in developing clinical applications for members of the TGF-b superfamily and their antagonists. For example, recombinant human BMP2 has been tested successfully in trials for the treatment of open tibial fractures [59]. In addition, topical application of TGF-b3 seems to be safe and beneficial in the treatment of pressure ulcers and is most effective at the earliest stages of therapy [60].
Clinical trials using activin or activin antagonists have yet to commence; however, the efficacy of these factors in animal disease models [51,52,57,58] suggests that they might also prove beneficial in treating human diseases. Follistatin and the activin Met108Ala receptor antagonist are particularly attractive drug candidates. Both are capable of differentially antagonizing several ligands of the TGF-b superfamily and thus could be useful therapeutic agents in tissue-specific settings.

Acknowledgements
This work was supported by a CJ Martin Fellowship from the National Health and Medical Research Council, Australia, to C.A.H.

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