Reprint of: Betaglycan: A multifunctional accessory

Molecular and Cellular Endocrinology 359 (2012) 13–22

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Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce

Reprint of: Betaglycan: A multifunctional accessory q Maree Bilandzic ⇑, Kaye L. Stenvers Prince Henry’s Institute, P.O. Box 5152, Clayton, Victoria 3168, Australia

a r t i c l e

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Article history: Received 23 December 2010 Received in revised form 18 April 2011 Accepted 18 April 2011 Available online 13 April 2012 Keywords: Betaglycan TGFb receptor III Inhibin

a b s t r a c t Betaglycan is a co-receptor for the TGFb superfamily, particularly important in establishing the potency of its ligands on their target cells. In recent years, new insights have been gained into the structure and function of betaglycan, expanding its role from that of a simple co-receptor to include additional ligand-dependent and ligand-independent roles. This review focuses on recent advances in the betaglycan field, with a particular emphasis on its newly discovered actions in mediating the trafficking of TGFb superfamily receptors and as a determinant of the functional output of TGFb superfamily signalling. In addition, this review encompasses a discussion of the emerging roles of the betaglycan/inhibin pathway in reproductive cancers and disease. Ó 2011 Elsevier Ireland Ltd. All rights reserved.

Betaglycan was originally identified as a non-signalling coreceptor for Transforming Growth Factor-b (TGFb), and its main function was defined as presenting ligand to the TGFb signalling receptors (López-Casillas et al., 1991; Wang et al., 1991). Subsequently, betaglycan was also shown to bind a distantly related member of the TGFb superfamily, inhibin A, regulating its binding to activin type II receptors (Lewis et al., 2000). In the last decade, additional ligand-dependent and -independent functions for betaglycan have been described which extend far beyond its role as a simple inhibin or TGFb accessory receptor (Wiater and Vale, 2003; Kirkbride et al., 2008; Lee et al., 2009; Mythreye and Blobe, 2009; Looyenga et al., 2010; Webber et al., 2010). Notably, betaglycan is an important regulator of reproduction (Sarraj et al., 2007; Escalona et al., 2009; Wiater et al., 2009; Glister et al., 2010) and fetal development (Stenvers et al., 2003; Compton et al., 2007; Sarraj et al., 2007, 2010; Walker et al., 2011) and is a tumor suppressor in many tissue types (Dong et al., 2007; Hempel et al., 2007; Sharifi et al., 2007; Turley et al., 2007; Finger et al., 2008a; Gordon et al., 2008; Bilandzic et al., 2009; Mythreye and Blobe, 2009; Stenvers and Findlay, 2010). The current literature indicates that betaglycan has complex roles in vivo, broadly influencing the activities and interactions of a number of TGFb superfamily members, thereby impacting diverse cellular processes. This review focuses on recent advances in our understanding of betaglycan structure and function, with a particular emphasis on its impact on inhibin and TGFb action. q A publishers’ error resulted in this article appearing in the wrong issue. The article is reprinted here for the reader’s convenience. For citation purposes, please use the original publication details; Molecular and Cellular Endocrinology 339 (2011) 180–189. DOI of original item: doi: 10.1016/j.mce.2011.04.014 ⇑ Corresponding author. Tel.: +61 3 9594 4372; fax: +61 3 9594 6125. E-mail address: [email protected] (M. Bilandzic).

0303-7207/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mce.2012.03.020

1. Betaglycan as a TGFb superfamily co-receptor The TGFb superfamily is a large group of structurally related growth factors, which includes the TGFbs, activins, inhibins, Bone Morphogenetic Proteins (BMPs), and Growth and Differentiation Factors (GDFs). These factors take part in the regulation of multiple cellular processes, including cell survival, proliferation, migration, and differentiation. As such, the superfamily is important for normal cellular function and turnover both during fetal development and in adult tissues. The actions of nearly all members of the superfamily are mediated by pairs of serine/threonine kinase receptors, the type I and II receptors, which form heteromeric complexes on the cell surface (Fig. 1). Ligands bind to their respective type I and II receptors causing a cascade of phosphorylation events and the activation of specific downstream signalling molecules. In the canonical pathway, TGFb superfamily members activate members of the SMAD transcription factor family (Fig. 1). Activins and TGFbs signal via SMAD2 and SMAD3 whereas BMPs signal via SMAD1/5/8. Receptor-activated SMADs then associate with the common SMAD4 and translocate to the nucleus to modify gene transcription (for review, see Hill, 2009). TGFbs can also activate members of the Mitogen Activated Protein (MAP) kinase signalling molecules, including JNK, p38, and ERKs, and the PI3 K/Akt pathway (for review, Ikushima and Miyazono, 2010). In addition to the kinase receptors, a number of other membrane-bound proteins participate in TGFb superfamily ligand binding and signalling. Of these, betaglycan is the most abundant TGFb receptor in many cell types (Segarini et al., 1989; Cheifetz et al., 1990). Betaglycan was originally identified as an accessory receptor for the TGFbs and is formally known as the type III TGFb receptor (TGFBR3) (López-Casillas et al., 1991; Wang et al., 1991). As betaglycan lacks a signalling domain and exhibits a slightly

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Fig. 1. TGFb superfamily signalling pathway. The diverse actions of the TGFb superfamily of growth and differentiation factors are mediated by pairs of serine-threonine kinase type I and type II receptors. In the canonical signalling pathway, agonists bind to specific sets of type I and type II receptors on the cell surface, which results in the phosphorylation and activation of downstream signalling molecules, the receptor regulated (R-)SMADs. Activated R-SMADs then associate with the common co-SMAD, SMAD4, and translocate to the nucleus where, in combination with cell-type specific binding partners, they regulate gene transcription and cellular function. The membranebound form of the accessory receptor, betaglycan, increases the binding of its ligands, the TGFbs, inhibins, and BMPs, to their signalling receptors. This generally enhances the signalling of TGFbs and BMPs and enhances the ability of inhibins to block the functions of other TGFb superfamily members. See text for details.

lower affinity for TGFbs than the type I/II receptor complex, the main function of this receptor was thought to be to bind TGFbs and present them to the type II receptor (López-Casillas et al., 1991; Wang et al., 1991). In accordance with this role, the presence of betaglycan on the cell surface increases the binding of the TGFbs to their type II receptors and increases ligand efficacy in biological assays (López-Casillas et al., 1993, 1994; Stenvers et al., 2003; Bilandzic et al., 2009). This effect is most pronounced for TGFb2, which binds poorly to the TGFb type II receptor in the absence of betaglycan (López-Casillas et al., 1993, 1994). In addition to binding TGFbs, betaglycan binds to inhibins with high affinity (Lewis et al., 2000) and is a major determinant of inhibin potency on pituitary gonadotrope cells (Escalona et al., 2009; Wiater et al., 2009). Within the TGFb superfamily, inhibins are unique as dedicated signalling receptors have yet to be reported. However, inhibins can bind to the type II receptors of other superfamily members (Lewis et al., 2000; Wiater and Vale, 2003). Notably, inhibins are closely related to activins. Mature activins (activin A, activin B, and activin AB) are disulfide-linked homoor heterodimers of two b subunits (bAbA, bBbB, and bAbB) while inhibins (inhibin A and inhibin B) are heterodimers of a- and bsubunits (abA, abB). Inhibins are capable of binding type II activin receptors through their b-subunits and functionally antagonizing activins by preventing the recruitment of activin type I receptors. However, inhibins can only bind with low affinity to the activin type II receptors, and are therefore not efficient competitors on their own (Lewis et al., 2000). For high potency inhibin action, the presence of betaglycan is required (Lewis et al., 2000; Escalona et al., 2009; Wiater et al., 2009). Betaglycan forms a stable complex with inhibin and type II activin receptors, thus sequestering activin type II receptors and reducing their availability to propagate activin signalling (Lewis et al., 2000). In a similar fashion, inhibin A

also antagonizes the binding of BMPs to activin and BMP type II receptors in the presence of betaglycan, resulting in the inhibition of BMP function (Wiater and Vale, 2003; Farnworth et al., 2006a). In addition, BMP-2, -4, -7 and GDF-5 have recently been shown to bind directly to the core domain of betaglycan, which may impact on both BMP receptor signalling and trafficking (see below; Kirkbride et al., 2008; Lee et al., 2009). As betaglycan can bind to several classes of TGFb superfamily ligands, it is not surprising that the functional impact of this receptor appears to be both context- and cell-type dependent. Indeed, as TGFbs, inhibins, and BMPs can be produced simultaneously by the same tissues, these ligands may compete at betaglycan to functionally antagonize each other (Ethier et al., 2002; Wiater et al., 2006; Farnworth et al., 2007; Kirkbride et al., 2008; Bilandzic et al., 2009; Looyenga et al., 2010). For example, there is significant overlap in the binding sites for TGFbs and inhibins within the membraneproximal domain of betaglycan (discussed below) (Wiater et al., 2006). The TGFbs have a higher affinity for betaglycan than inhibins, and neither inhibin A nor inhibin B are able to compete for betaglycan binding sites against [125I]TGFb1 or [125I]TGFb2 in gonadal or adrenal cell lines (Farnworth et al., 2007; Looyenga et al., 2010). The functional impact of this has been demonstrated in murine LbT2 gonadotrope cells, in which TGFbs can block the access of inhibin A to betaglycan, thereby relieving the inhibinmediated antagonism of activin responses (Ethier et al., 2002). Other mechanisms may also come into play as TGFb1 and TGFb2 also down-regulate the expression of the betaglycan gene in gonadal or adrenal cell lines and therefore may also indirectly reduce cellular sensitivity to inhibins in this manner (Farnworth et al., 2007). Furthermore, in some contexts, inhibins or BMPs may also inhibit TGFbs via betaglycan (Kirkbride et al., 2008; Looyenga et al., 2010). To what degree competition occurs between betagly-

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can ligands in vivo is yet unclear, but the in vitro data suggest that betaglycan might integrate multiple TGFb superfamily inputs into discrete functional outcomes. Emerging data indicate that this occurs not only via betaglycan’s role in ligand presentation, but also through its distinct effects on the receptor trafficking and downstream signalling of each of its ligand subtypes, which are discussed further below. 2. Emerging features of betaglycan structure 2.1. Ectodomain Betaglycan is an 851 amino acid proteoglycan, comprising a large extracellular domain, a single-pass transmembrane region, and a short 42 amino acid intracellular domain (Fig. 2) (López-Casillas et al., 1991, 1994; Wang et al., 1991). Betaglycan exists on the cell surface as noncovalently linked homodimers (Mendoza et al., 2009). Mutagenesis studies have identified two non-overlapping ligand binding regions within the extracellular domain, an aminoterminal binding site and a membrane-proximal binding site (López-Casillas et al., 1991, 1994; Pepin et al., 1994; Esparza-López et al., 2001; Mendoza et al., 2009). The two binding domains fold independently of one another and can independently function to present ligand to type II receptors (Esparza-López et al., 2001). The two binding domains are joined by an unstructured 50-amino acid linker region, which contains a proteolytic cleavage site (Mendoza et al., 2009). Betaglycan binds all three TGFbs, inhibin A, inhibin B, and certain BMPs via its extracellular domain (Lewis et al., 2000; Wiater et al., 2006; Kirkbride et al., 2008). The TGFbs and BMPs bind to both the membrane-distal and membrane-proximal binding domains, with TGFb2 having higher affinity than other ligands (López-Casillas et al., 1991, 1994; Wang et al., 1991; Wiater et al., 2006; Kirkbride et al., 2008; Mendoza et al., 2009). In contrast, inhibin A and inhibin B bind only to the membrane-proximal binding domain (Wiater et al., 2006; Makanji et al., 2008). Mutagenesis studies have shown that there is considerable overlap in ligand

Fig. 2. Structure of betaglycan. Structural features of the full-length form of betaglycan, depicting key binding domains, enzymatic cleavage sites, and intracellular residues important for the association of adaptor proteins. Proteolytic cleavage events and differential post-translational modification of the GAG sidechains in specific cellular contexts results in the generation of multiple forms of betaglycan, the functions of which are poorly understood. MT1-MMP, MT3-MMP, or a related protease can cleave the ectodomain near the transmembrane region to generate a soluble receptor, capable of binding ligand. Plasmin has been shown to cleave the linker region of the soluble receptor, generating two fragments with reduced ability to bind ligand. See text for details.

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binding sites within the membrane-proximal domain, with residues 608–620 essential for both inhibin and TGFb binding, although specific amino acid residues contribute differentially to the binding of each class of growth factor (Wiater et al., 2006). Inhibin A and inhibin B appear to have different requirements for betaglycan (Makanji et al., 2008, 2009). Inhibin A exhibits a higher affinity than inhibin B for betaglycan/activin type II receptor complexes (Makanji et al., 2009) and can inhibit BMP responses via betaglycan in rodent adrenal cells while inhibin B cannot (Farnworth et al., 2006a). Nevertheless, inhibin B more potently antagonizes Follicle Stimulating Hormone (FSH) release from pituitary gonadotropes, indicating that inhibin B may utilize additional binding proteins in this cell type to achieve high affinity binding to activin type II receptors (Makanji et al., 2009). The extracellular domain also contains two glycosaminoglycan (GAG) attachment sites for the addition of heparan and chondroitin sulfate GAG side chains, located at Ser535 and Ser546 (Cheifetz et al., 1988; López-Casillas et al., 1994). The GAG side chains are not essential for ligand binding to the core protein (López-Casillas et al., 1993; Wiater et al., 2006) but may mediate binding to other classes of growth factors, such as the fibroblast growth factors (Cheifetz et al., 1988; Andres et al., 1992; López-Casillas et al., 1994). The GAG side chains appear to contribute to the function of betaglycan as mutation of the GAG attachment sites reduces betaglycan-mediated inhibition of migration (Mythreye and Blobe, 2009). In addition, the GAG side chains may block ligand access to the signalling receptors in some cell types, thus disrupting TGFb signalling and function (Eickelberg et al., 2002). Adding to the complexity in betaglycan action is the fact that its ectodomain can be regulated by proteolytic cleavage near the transmembrane domain, which generates a soluble 120 kDa form of the receptor (Velasco-Loyden et al., 2004). Soluble betaglycan is found in serum, milk, and the extracellular matrix (Andres et al., 1989; Cheung et al., 2004). Soluble betaglycan has distinct functions from the membrane bound form, with the soluble form proposed to sequester ligands from their signalling receptors, thereby antagonizing signalling (López-Casillas et al., 1994). Despite its clear importance as a determinant of betaglycan function, little is understood about the regulation of betaglycan cleavage at the cell membrane. Treatment with pervanadate, a tyrosine phosphatase inhibitor, generates the 120 kDa fragment corresponding to the entire betaglycan ectodomain plus a novel 90 kDa fragment, each still capable of binding TGFbs (Velasco-Loyden et al., 2004). Overexpression of two membrane type matrix metalloproteases (MMPs), MT1-MMP and MT3-MMP, indicate that these or a related protease can generate the 90 kDa fragment (Velasco-Loyden et al., 2004). Binding of TGFb ligands by the soluble receptor is affected by cleavage of the 50-amino acid linker region in the ectodomain (Mendoza et al., 2009). Plasmin-mediated cleavage of soluble betaglycan within this linker region generates fragments comprising the two binding domains of 45 and 55 kDa, which can no longer dimerize and exhibit a reduced ability to bind and neutralize TGFbs (Mendoza et al., 2009). The transmembrane-cytoplasmic fragment that remains following ectodomain shedding has recently been shown to undergo cleavage by the intramembrane protease c-secretase to release the cytoplasmic fragment (Blair et al., 2011). This cleavage event appears to be an important determinant of betaglycan function, as the free cytoplasmic domain may both contribute to TGFb signalling and exhibit novel functions within the cell (Blair et al., 2011). However, the regulatory mechanisms that govern the various types of betaglycan cleavage events in different physiological and pathological settings are largely unknown. Furthermore, as nearly all work on betaglycan cleavage products has been done in relation to the actions of TGFbs, it is yet unclear how soluble and cytoplasmic fragments of betaglycan affect BMP and inhibin function.

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2.2. Cytoplasmic domain

3.1. Regulation of TGFb superfamily receptor trafficking

Full-length betaglycan contains a short cytoplasmic tail which does not exhibit kinase activity nor is it required for betaglycan’s ligand presentation role (López-Casillas et al., 1991; Wang et al., 1991; Esparza-López et al., 2001). However, the cytoplasmic domain is highly conserved between species and has been shown to greatly impact betaglycan function. Specifically, sequences have been identified within the cytoplasmic tail of betaglycan that allow for association with adaptor proteins. The last three amino acids in the C-terminus of betaglycan comprise a class I PDZ binding motif and have been shown to bind with GAIP-interacting protein, C-terminus (GIPC) (Blobe et al., 2001). This interaction stabilizes endogenous or stably expressed betaglycan at the cell membrane and increases TGFb responsiveness in Mv1Lu mink lung epithelial cells and L6 myoblasts (Blobe et al., 2001). More recently it has been shown that GIPC-betaglycan interaction can also inhibit TGFbmediated SMAD signalling and migration in breast cancer cells, suggesting that the actual functional outcome of the GIPC interaction depends on cell type and/or context (Lee et al., 2010). The exact mechanisms by which the GIPC-betaglycan interaction results in distinct functional consequences in different cell types are as yet incompletely understood but may point to the existence of additional adaptor proteins or binding partners that modify downstream functional outcomes. Another important intracellular protein interaction is the association of the scaffolding protein b-arrestin2 with betaglycan. Arrestins were originally identified as regulators of G protein-coupled receptors which bind to activated receptors, targeting them for internalization and desensitization (Kovacs et al., 2009). Arrestins have since been shown to associate with a number of other cell surface receptors and mediate a variety of distinct downstream outcomes, including mediating receptor turnover and potentiating particular signalling pathways (Kovacs et al., 2009). The interaction between b-arrestin2 and betaglycan is both ligandand GAG-independent but requires betaglycan to be phosphorylated at threonine (Thr) 841 by the type II TGFb receptor (Chen et al., 2003). As a result of the b-arrestin2-betaglycan association, betaglycan and the type II receptor are co-internalized via a clathrin-independent/lipid raft pathway, and TGFb signalling is suppressed (Chen et al., 2003; Finger et al., 2008b). The association of betaglycan with b-arrestin2 also results in the internalization of ALK6 type I BMP receptor, conversely, enhancing BMP signalling (Lee et al., 2009). Via its interaction with b-arrestin2, betaglycan influences a number of signalling pathways, including both SMAD-dependent and -independent signalling (You et al., 2007; Finger et al., 2008a; Mythreye and Blobe, 2009). As arrestins are known to scaffold receptors to particular signalling pathways (Kovacs et al., 2009), the association of betaglycan with b-arrestin2 may be a determinant of which signals arise downstream of betaglycan-type II receptor complexes. The functional implications of the betaglycan-b-arrestin2 interaction will be discussed further below.

The exact functional response elicited by specific growth factors of the TGFb superfamily in any particular cell type depends in part on the strength and duration of the downstream signals. The magnitude of downstream signalling is determined by a variety of factors which include not only ligand concentration but the availability and stability of the cohort of receptors and modulating binding proteins on the cell surface (Jullien and Gurdon, 2005). The latter is governed by receptor turnover, which occurs via internalization of the receptors, and routing to the endosomes for recycling, degradation, and/or the propagation of signals. The selection of the routing pathway which determines the subcellular localization and fate of the receptors is not entirely understood but is thought to be affected by various cell surface and intracellular protein interactions (for review see, Chen, 2009; Kardassis et al., 2009; Kovacs et al., 2009; Lönn et al., 2009). TGFb receptors are internalized in a constitutive fashion independent of ligand binding. TGFb receptors can be internalized by either the clathrin-dependent pathway or a clathrin-independent/lipid raft and caveolin-mediated pathway. In the clathrin-dependent pathway, TGFb receptors are internalized in clathrin-coated pits, routed into early endosomes, and recycled back to the cell surface. This pathway is linked to the activation of SMAD-dependent signalling, with the endosomal membranes thought to serve as signalling platforms (Chen, 2009). In contrast, TGFb receptors associated with lipid rafts are routed towards a degradative pathway, by routing the receptors into caveolar vesicles where they are targeted for ubiquitination and degradation. Routing of activin and BMP receptors into particular intracellular compartments is also thought to determine the strength and duration of their signalling (Jullien and Gurdon, 2005; Kardassis et al., 2009) although it is not yet clear that the same mechanisms regulate the internalization of all TGFb superfamily receptors (Kardassis et al., 2009). In fact, there appears to be considerable cell-type specificity in the mechanisms of TGFb superfamily receptor internalization and the resulting signalling output (Kardassis et al., 2009). Similar to the signalling receptors, the route of endocytosis differentially affects the fate of betaglycan. Betaglycan colocalizes with markers of clathrin-dependent (SARA, Rab9, clathrin) and clathrin-independent internalization (caveolin, flottilin) (Finger et al., 2008b), indicating that, like the type I and II TGFb receptors, betaglycan is internalized via both pathways. The clathrin-independent pathway downregulates betaglycan from the cell surface and is necessary for both signalling and receptor degradation (Finger et al., 2008a). Although the cytoplasmic domain of betaglycan is not strictly required for the receptor internalization, a mutant form of betaglycan lacking the cytoplasmic domain is internalized at a significantly slower rate (Finger et al., 2008b). This is at least partially dependent on the association of the cytoplasmic tail of betaglycan with b-arrestin2 (Chen et al., 2003; Finger et al., 2008a). The mechanisms underlying betaglycan trafficking in particular cell types and the distinct functional outcomes on each endocytic pathway are still emerging (Fig. 3). The current data indicate that betaglycan can influence the fate of the signalling receptors for each of its ligands. Notably, betaglycan has been recently shown to direct the type I and II TGFb receptors towards clathrin-mediated internalization and into early endosomes, increasing the stability of the type I/II receptor complex and basal TGFb signalling levels in HEK293T and HepG2 cells (McLean and Di Guglielmo, 2010). This effect occurred independently of ligand stimulation, and both the type I and II receptors could stably interact with betaglycan and be trafficked into non-lipid raft fractions independently of one another (McLean and Di Guglielmo, 2010). Intriguingly, the binding of different classes of ligands to betaglycan may redirect how betaglycan is trafficked in the cell. In adrenocortical cells,

3. Emerging functions for betaglycan As mentioned above, in recent years, additional cellular functions of betaglycan have been identified, expanding on its role as a simple TGFb or inhibin co-receptor to include broader roles as a modulator of the activities of multiple ligands of the TGFb superfamily. In addition, it is now apparent that betaglycan may also have ligand-independent roles. We discuss below the growing complexity of betaglycan function, with particular emphasis on its role in receptor trafficking and its impact on downstream signalling.

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Fig. 3. Intracellular trafficking of betaglycan. Emerging data indicate that the binding of different ligands to betaglycan differentially affects its subcellular routing and fate. (A) The type I and II TGFb receptors can be internalized by either the clathrin-dependent pathway, which leads to receptor recycling and downstream signalling, or a lipidraft-dependent pathway, which leads to receptor degradation. Via a b-arrestin2-dependent mechanism, the presence of betaglycan on the cell surface directs the type I and II TGFb receptors towards the clathrin-mediated pathway and enhances signalling (indicated by the relative thickness of the lines). This occurs both constitutively and upon TGFb stimulation. (B) If inhibin A binds to betaglycan instead, betaglycan is internalized via a novel pathway which is neither clathrin- or lipid-raft dependent, leading to temporary down-regulation of betaglycan on the cell surface, desensitization of cells to TGFbs, and eventual recycling of betaglycan to the cell surface.

while TGFb2 resulted in the internalization of betaglycan via a clathrin-dependent mechanism, the mode of inhibin A-mediated internalization was neither clathrin- nor lipid raft/caveolin-dependent (Looyenga et al., 2010). The TGFb2-mediated route resulted in the degradation of betaglycan, as the total level of betaglycan expression was reduced. In contrast, inhibin A resulted in the down-regulation of betaglycan at the cell surface but did not affect total betaglycan levels, suggesting that betaglycan was recycled rather than degraded following inhibin binding (Looyenga et al., 2010). These findings are significant because they indicate that inhibin antagonizes TGFb superfamily ligands in more ways than just simply binding to betaglycan and the relevant type II receptors. In addition, these data provide a novel mechanism by which TGFb function can be suppressed by inhibin A, which has a lower affinity for betaglycan and cannot compete off bound TGFb (Farnworth et al., 2007; Looyenga et al., 2010). It has not yet been determined whether betaglycan is internalized following inhibin binding in a complex with activin or BMP type II receptors. However, betaglycan has recently been shown to differentially mediate trafficking of the BMP type I receptors, ALK3 and ALK6 (Lee et al., 2009). Betaglycan mediated ALK6 internalization in a b-arrestin2-dependent fashion, leading to an increase in SMAD1 activation (Lee et al., 2009). In contrast, ALK3 was retained at the cell surface by a betaglycan-dependent mechanism (Lee et al., 2009). Collectively, these studies indicate that betaglycan differentially dictates the trafficking of the signalling receptors for its various ligands, thus influencing receptor fate and signalling output. 3.2. Regulation of TGFb superfamily signalling output Different concentrations of ligand alter the functional outcomes achieved by TGFb superfamily action. For example, in cultured murine podocytes, autocrine TGFb2 induces growth arrest and differentiation via a SMAD3-dependent pathway while higher concentrations of TGFb activate both SMAD3 and p38 MAPK pathways, resulting in cell death (Wu et al., 2005). As discussed above, through its roles in ligand presentation and the regulation of sig-

nalling receptor trafficking, betaglycan is a major determinant of cellular responsiveness to TGFb superfamily members and, as such, influences the functional consequences of their action. In general, betaglycan can inhibit or enhance the signalling of particular TGFb superfamily members via cell type/contextdependent mechanisms which are still poorly understood. For example, membrane-bound betaglycan enhances TGFb signalling via SMAD2 and SMAD3 but, by way of inhibin binding, opposes activin signalling by these same transcription factors (Lewis et al., 2000; Bilandzic et al., 2009). In addition, in certain cell types, betaglycan appears to differentially regulate SMAD pathway activation and hence direct specific downstream transcriptional responses. In L6 myoblasts, betaglycan enhances TGFbmediated growth arrest via the selective activation of the SMAD3 and p38 MAPK pathways, with no effect on the SMAD2 pathway (You et al., 2007). In these cells, betaglycan was equally effective in enhancing TGFb1- and TGFb2-mediated responses, suggesting that its effects on signalling were not simply due to its ligand presentation role, as TGFb1 does not require betaglycan to bind to the type II receptor with high affinity (You et al., 2007). Recent work may shed light on the underlying mechanism by which betaglycan influences TGFb1 signalling. Exosomes are secreted vesicles that mediate communication between cells (Denzer et al., 2000). Exosomes secreted by some cancer cell types express TGFb1, which drives the differentiation of cancer-associated fibroblasts into myofibroblasts, which in turn promotes tumor growth and spread (Webber et al., 2010). In a novel mechanism of action, betaglycan was found to tether TGFb1 to the surface of cancer cell exosomes, where it stimulated SMAD3 activation (Webber et al., 2010). Exosomal betaglycan expression was required for TGFb1mediated myofibroblast differentiation (Webber et al., 2010), indicating that betaglycan determined TGFb1 function in these cells. As betaglycan also enhances TGFb-mediated SMAD2 signalling and BMP-mediated SMAD1 signalling in certain cell types (Stenvers et al., 2003; Lee et al., 2009), this previously unknown function of betaglycan may also contribute to the activation of other SMAD-mediated pathways.

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3.3. Regulation of SMAD-independent pathways In addition to SMAD signalling pathways, betaglycan is also known to enhance or inhibit SMAD-independent signalling, although it remains to be determined how specific pathways are differentially influenced downstream of betaglycan in different cellular contexts. Several studies indicate that betaglycan can impact the function of nuclear factor kappa B (NFjB), a transcription factor which regulates genes involved in inflammation, immune response, and apoptosis (Criswell and Arteaga, 2007; Criswell et al., 2008; You et al., 2009). NFjB is sequestered in an inactive state in the cytoplasm, bound to inhibitory kappa B protein (IjB). Upon ligand activation, IjB is phosphorylated by an IjB kinase, resulting in IjB degradation and the activation of NFjB. Silencing betaglycan gene expression in MCF10A breast epithelial and MDA-MB-231 breast cancer cells results in IjB degradation and NFjB-mediated transcriptional activation by a b-arrestin2-dependent mechanism (You et al., 2009). Betaglycan blocked NFjB signalling both with and without TGFb stimulation, indicating that the inhibition of NFjB signalling may be a function separate to betaglycan’s TGFb-binding role in these cell lines. Similarly, silencing betaglycan in nontumorigenic NMuMG mouse cells increased cell motility and proliferation in an in vivo xenograft mouse model, which was associated with increased NFjB-mediated gene transcription and downregulation of E-cadherin (Criswell and Arteaga, 2007). However, betaglycan’s effects on NFjB activity appear to be cell type and/or context dependent as knockdown of endogenous betaglycan expression in MDA-231 human breast cancer cells or mouse mammary cancer cells reduced their metastatic potential and NFjB activity, indicating that betaglycan enhances NFjB activity in these particular cell lines (Criswell et al., 2008). This is reminiscent of the complexity of TGFb’s crosstalk with the NFjB pathway, as TGFb can either counter or contribute to NFjB-mediated responses in particular cell types, and the mechanisms underlying the switch in functional outcomes are largely unknown (Criswell et al., 2008). Emerging data indicate that betaglycan also regulates other signalling pathways in a complex manner. In L6 myoblasts, the presence of betaglycan resulted in p38 MAPK pathway activation in a TGFb/type I receptor-dependent manner (You et al., 2007). However, in a renal cancer cell line, betaglycan expression similarly resulted in p38 activation but in a TGFb/type II receptor-independent manner (Margulis et al., 2008). These effects depend in part on an intact cytoplasmic domain (You et al., 2007; Margulis et al., 2008), pointing to a role for the adaptor proteins associated with the cytoplasmic tail of betaglycan. Betaglycan can also activate the small Rho GTPase Cdc42 in ovarian epithelial cells via a TGFb/type I receptor independent pathway, and this activation was important for the anti-migratory effects of betaglycan (see below) (Mythreye and Blobe, 2009). Whether the betaglycan-mediated activation of SMAD-independent pathways are truly ligandindependent or due to one of the ligands that do not signal through the type I and II TGFb receptors is yet unclear. The activation of Cdc42 by betaglycan may also be influenced in part by its GAG side chains as the downstream functional effects on ovarian cell migration were reduced in betaglycan mutants lacking the GAG attachment sites (Mythreye and Blobe, 2009).

of literature indicates that betaglycan plays essential, non-redundant roles in fetal development and reproduction (Brown et al., 1999; Stenvers et al., 2003; Compton et al., 2007; Sarraj et al., 2007, 2010; Walker et al., 2011) and links disruption in betaglycan expression or function to multiple pathologies (Florio et al., 2005; Dixit et al., 2006; Chand et al., 2007b; Dong et al., 2007; Hempel et al., 2007; Sharifi et al., 2007; Turley et al., 2007; Finger et al., 2008b; Gordon et al., 2008; Bilandzic et al., 2009; Zhu et al., 2009; Cooper et al., 2010). Notably, many reproductive and endocrine tissues co-ordinately express betaglycan in conjunction with specific sets of TGFb superfamily type I and II receptors and in some tissues, betaglycan’s function has been determined relative to particular classes of ligands (Copland et al., 2003; Dong et al., 2007; Hempel et al., 2007; Gordon et al., 2008; Margulis et al., 2008; Bilandzic et al., 2009). However, generally, due to its promiscuous binding nature and complex modes of action, the exact mechanisms by which betaglycan acts in particular physiological settings are unknown. We discuss below the best-established roles for betaglycan in reproductive and endocrine organ function and disease, with a particular emphasis on organs in which roles of the betaglycan/inhibin pathway have been demonstrated in multiple model systems. For more comprehensive coverage of particular endocrine organs, the reader is referred to the other detailed reviews within this special issue of Molecular and Cellular Endocrinology. 4.1. Pituitary Mounting evidence indicates that betaglycan is required for inhibins, particularly inhibin A, to act on gonadotropes and other target cells (Farnworth et al., 2007; Makanji et al., 2008, 2009; Escalona et al., 2009; Wiater et al., 2009). Inhibins are produced by the somatic cells of the gonads and act in an endocrine manner on pituitary gonadotropes to regulate the secretion of FSH: activins stimulate FSH release while inhibins oppose this action (Ling et al., 1986; Vale et al., 1986). In turn, FSH acts back on the gonads to regulate diverse cellular processes, particularly in the somatic cell lineages, which are the essential support cells for the developing sperm and oocytes. Recent studies using betaglycan gene knockdown in the mouse LbT2 pituitary gonadotrope cell line demonstrated betaglycan is a key determinant of inhibin efficacy in the anterior pituitary (Wiater et al., 2006; Escalona et al., 2009). Notably, betaglycan gene knockdown impaired the ability of inhibin A to antagonize activin-stimulated FSH production and gonadotropin releasing hormone receptor promoter activity (Escalona et al., 2009; Wiater et al., 2009). The role of betaglycan as an inhibin co-receptor may be particularly important for the regulation of the reproductive hormone axis in both males and females, as evidenced by the large number of reproductive disorders associated with alterations in betaglycan or inhibin expression in human and rodent models (Matzuk et al., 1992; Boggess et al., 1997; Stenvers et al., 2003; Kumanov et al., 2005; Robertson and Oehler, 2005; Steller et al., 2005; Dixit et al., 2006; Hamar et al., 2006; Chand et al., 2007a,b; Dong et al., 2007; Hempel et al., 2007; Mom et al., 2007; Sarraj et al., 2007; Sharifi et al., 2007; Tsigkou et al., 2007,2008a,b; Turley et al., 2007; Balanathan et al., 2009; Bilandzic et al., 2009; Mythreye and Blobe, 2009; Zhu et al., 2009; Sarraj et al., 2010; Stenvers and Findlay, 2010).

4. Impact of betaglycan on biology

4.2. Gonads

TGFb superfamily members play major roles in the control of growth, differentiation, death, and migration of most cell types. As a widely expressed accessory receptor for multiple classes of growth factors within the superfamily, betaglycan has the potential to influence diverse cellular processes. Indeed, a growing body

In addition to their role in the anterior pituitary, inhibins and betaglycan are also thought to have local actions in the gonads, in particular in the somatic cell populations (Stenvers and Findlay, 2010). In the ovary, betaglycan is first expressed as granulosa cells differentiate from their precursors (Sarraj et al., 2007), and betagly-

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can expression in cultured granulosa cells is upregulated by estrogen and gonadotrophins, the hormonal drivers of folliculogenesis (Liu et al., 2003; Omori et al., 2005). In addition, betaglycan is expressed in theca cells, and this expression increases as follicles grow and mature, suggesting that granulosa cell-derived inhibins may be able to exert greater regulation on theca during late folliculogenesis (Glister et al., 2010). Glister et al. (2010), demonstrated the functional impact of betaglycan on bovine thecal cells, showing that inhibin acts via betaglycan to antagonize BMP-mediated downregulation of Luteinizing Hormone-stimulated steroidogenesis. In accordance with the expression of betaglycan in follicular cells, alterations in betaglycan are associated with ovarian disorders in humans. Human granulosa cell tumors (GCTs) exhibit a loss of betaglycan and reduced sensitivity to both inhibins and TGFbs, which may contribute to the human disease (Bilandzic et al., 2009) (see below). In addition, premature ovarian failure in specific human populations is associated with mutations in INHA (inhibinalpha subunit) or in the shared TGFb/inhibin binding domain of betaglycan (Dixit et al., 2006; Chand et al., 2007a,b). In contrast, betaglycan expression was elevated and associated with alterations in circulating pituitary- and ovarian-derived hormones in ovaries from women with polycystic ovarian syndrome (Zhu et al., 2009). As follicular cell types also respond to other TGFb superfamily members in addition to inhibins, betaglycan likely also regulates the actions of its other ligands within the ovary. However, the exact roles of the TGFbs appear to highly species-dependent and variable across folliculogenesis, and it is not yet clear how betaglycan affects TGFb function in the ovary (Drummond et al., 2002, 2003; Knight and Glister, 2006). Nevertheless, the data point to key roles for betaglycan and its ligands in regulating normal folliculogenesis and preventing dysregulated follicular growth in humans. Similar to the ovary, in the mammalian testis, betaglycan is predominantly expressed by the somatic cells, which also express the inhibin a- and b-subunits (Lewis et al., 2000; Anderson et al., 2002; Sarraj et al., 2007). The localization of betaglycan to Sertoli and Leydig cells indicates potential autocrine or paracrine actions of betaglycan’s ligands on these cells types. Notably, adult Leydig cells also express a full complement of type I and type II TGFb and activin receptors, as well as their downstream SMAD effectors (Bernard, 2003; Itman et al., 2006; Loveland et al., 2007). Activins and TGFbs act in vitro to inhibit steroidogenesis in mammalian Leydig cells, resulting in decreased production of androgens (Hsueh et al., 1987; Mauduit et al., 1991; Le Roy et al., 1998, 1999; Olaso et al., 1999), and addition of exogenous inhibin to Leydig cell cultures blocks activin-mediated inhibition of testosterone production (Hsueh et al., 1987; Lin et al., 1989; Risbridger et al., 1989). Although a role for betaglycan in mediating its ligands’ actions on adult Leydig cells has yet to be directly demonstrated, betaglycan has been shown to be essential for normal testis development, in particular for the development of somatic cell populations during early gonadogenesis (Sarraj et al., 2010). Betaglycan is expressed by fetal Leydig cells in rodent and human testis (Anderson et al., 2002; Sarraj et al., 2007), and betaglycan null murine fetal testis exhibits disrupted Leydig cell differentiation, with reduced expression of several genes involved in somatic cell development and steroidogenesis (Sarraj et al., 2010). In addition, studies in the Leydig-like TM3 cell line indicate that betaglycan may be a site of competition between its ligands (Farnworth et al., 2007). Specifically, through their higher affinity for the shared TGFb-inhibin binding domain, the TGFbs directly block access of inhibins to betaglycan and also downregulate betaglycan expression by TM3 cells, thus reducing their sensitivity to inhibins by two different mechanisms (Farnworth et al., 2007).

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4.3. Adrenal Inhibin a- and b-subunits are expressed and may act within the adrenal gland to regulate steroidogenesis (Vanttinen et al., 2002, 2003). In a mouse adrenocortical cell line, activins and BMPs suppressed cytochrome P450 17a-hydroxylase 17,20-lyase (Cyp17a1) expression and the corresponding production of 17a-hydroxyprogesterone which arises from 17a-hydroxylase activity (Farnworth et al., 2006a). Inhibins blocked activin- and BMP-mediated suppression of Cyp17a1 in these cells (Farnworth et al., 2006a), which express betaglycan as well as additional, unidentified inhibin binding proteins (Farnworth et al., 2006b). Betaglycan and all other components of the activin/inhibin signalling system are also expressed in the adult human adrenal cortex, where betaglycan may be required to mediate inhibin’s antagonism of activin-mediated functions, such as inhibition of steroidogenesis and induction of apoptosis (Vanttinen et al., 2002, 2003). Disruption of betaglycan function may be involved in the development of certain adrenal diseases, in particular, cancers. Betaglycan and INHA genes are both downregulated in adrenal cancers (de Fraipont et al., 2005; Hofland et al., 2006). Furthermore, in gonadectomised Inha null mice, adrenocortical tumors may form due to the loss of inhibin’s antagonism of TGFb2, which occurs through the inhibin-mediated internalization of betaglycan (Looyenga et al., 2010). 4.4. Roles as a tumor suppressor in many endocrine and non-endocrine tissues In recent years, betaglycan has been identified as a tumor suppressor in many human cell types. In particular, loss of this receptor equates with a loss of sensitivity to TGFb and/or inhibin-mediated control of growth and cancer cell migration (Dong et al., 2007; Sharifi et al., 2007; Turley et al., 2007; Finger et al., 2008b; Gordon et al., 2008; Hempel et al., 2008; Bilandzic et al., 2009). Benign tissue is transformed into metastatic cancer through a series of steps, including the mutations in or epigenetic silencing of tumor suppressor genes (Bierie and Moses, 2010). Several recent studies demonstrated that cancer cells exhibit a down-regulation or loss of betaglycan expression at the mRNA and/or protein levels which correlates with increasing tumor progression. Loss of betaglycan expression occurs in cancers by a variety of mechanisms, including frequent loss of heterozygosity at the betaglycan gene locus and epigenetic silencing (Dong et al., 2007; Sharifi et al., 2007; Cooper et al., 2010). In addition, in renal carcinoma, loss of betaglycan expression is secondary to epigenetic silencing of GATA3, a key transcription factor which acts on the betaglycan promoter to drive expression (Cooper et al., 2010). The extent of ectodomain shedding may also shift the balance of soluble and cell-surface betaglycan expression in some cancers, thus altering the impact of betaglycan expression on different types or stages of tumorigenesis (Bernabeu et al., 2009). A functional role for soluble betaglycan has been demonstrated in human pancreatic cancer cells, in which soluble betaglycan suppresses cell motility by inhibiting the epithelial-tomesenchymal transition of tumor cells (Gordon et al., 2008). In some cases, the pathogenesis of specific cancer types has been linked to betaglycan’s function relative to particular classes of its ligands (Copland et al., 2003; Dong et al., 2007; Hempel et al., 2007; Gordon et al., 2008, 2009; Margulis et al., 2008; Bilandzic et al., 2009). For example, disrupted inhibin/betaglycan function is most strongly linked to ovarian cancers. The gene encoding the inhibin-a subunit (Inha) is a tumor suppressor in mice as Inha deletion results in granulosa/Sertoli cell tumors in the gonads and adrenals of both sexes (Matzuk et al., 1992). In humans, however, GCTs are generally not associated with a loss of INHA expression,

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but instead exhibit high inhibin expression (Fuller and Chu, 2004). Human GCTs have recently been demonstrated to exhibit reduced betaglycan expression compared to normal pre-menopausal ovary, suggesting that human GCTs may occur due to a loss of responsiveness to inhibins by ovarian tumor cells (Bilandzic et al., 2009). Furthermore, using ligand-specific binding mutants of betaglycan (Wiater et al., 2006) and INHA gene silencing in GCT cell lines, it was demonstrated that the loss of betaglycan on GCT cells blocked the inhibin-mediated negative regulation of GCT migration and invasion (Bilandzic et al., 2009). Similarly, in epithelial ovarian cancer cells, overexpression of betaglycan inhibited cell migration while INHA gene silencing enhanced both migration and invasion (Hempel et al., 2007). In a survey of ovarian epithelial cancer cell lines, the loss of inhibin A responsiveness in epithelial ovarian cancer cell lines was associated with a more aggressive tumor phenotype both in vitro and in an in vivo xenograft model, suggesting that the loss of betaglycan/inhibin function may be a common occurrence in ovarian cancers (Steller et al., 2005). Indeed, loss of betaglycan expression was found in 53% of GCTs (Bilandzic et al., 2009) and 63–86% of ovarian epithelial cancers, with increasing loss of betaglycan associated with more advanced disease (Hempel et al., 2007). Mechanistically, loss of betaglycan from tumor cells is frequently associated with increased cell migration and invasion, increased angiogenesis, and increased tumor progression (Gatza et al., 2010). In particular, betaglycan inhibited both random and directional migration of ovarian cancer cells via the activation of the small GTPase Cdc42 in a b-arrestin2-dependent fashion, which was associated with reorganization of the actin cytoskeleton and the appearance of filopodial structures (Mythreye and Blobe, 2009). This function of betaglycan was independent of TGFb stimulation, which suggests that inhibins may act via betaglycan instead to regulate motility in ovarian epithelial cells. This pathway may play a similar role in other inhibin target tissues such as adrenal (Looyenga et al., 2010) and endometrium (Florio et al., 2005), although generally betaglycan’s role in mediating inhibin action in these tissues is not well-defined. Betaglycan also appears to act as a tumor suppressor in many other tissues, as well, broadly inhibiting the development of metastatic features; most often, this role has been linked to betaglycan’s role as a TGFb accessory receptor (extensively reviewed elsewhere, see Gatza et al., 2010; Stenvers and Findlay, 2010). However, in ovarian granulosa cells, overexpression of betaglycan in tumor cells lacking its expression results in the restoration of both TGFb and inhibin responses, which collectively regulate cell-substrate adhesion, wound healing, migration, and invasion (Bilandzic et al., 2009). These data suggest that betaglycan may be tumor suppressive via the integration of multiple TGFb superfamily signals in a single cell type.

5. Concluding remarks The current literature indicates that betaglycan plays essential, non-redundant roles in fetal and adult health, both enhancing and inhibiting the actions of its multiple ligands in a context-dependent manner. However, the regulation of ectodomain shedding of betaglycan and the collective impact of the various membranebound and soluble forms of betaglycan in most physiological and pathological settings remain poorly understood. Given the complex nature of betaglycan structure and function, it will be important to determine the importance of betaglycan to particular cellular and disease processes. Additional studies into betaglycan’s structure/function are required to clarify whether betaglycan expression within tissues is diagnostic of certain diseases, such as cancers. This work was supported by the Victorian Government’s Operational Infrastructure Support Program.

References Anderson, R.A., Cambray, N., Hartley, P.S., McNeilly, A.S., 2002. Expression and localization of inhibin alpha, inhibin/activin betaA and betaB and the activin type II and inhibin beta-glycan receptors in the developing human testis. Reproduction 123, 779–788. Andres, J., DeFalcis, D., Noda, M., Massagué, J., 1992. Binding of two growth factor families to separate domains of the proteoglycan betaglycan. J. Biol. Chem. 267, 5927–5930. Andres, J.L., Stanley, K., Cheifetz, S., Massague, J., 1989. Membrane-anchored and soluble forms of betaglycan, a polymorphic proteoglycan that binds transforming growth factor-beta. J. Cell Biol. 109, 3137–3145. Balanathan, P., Williams, E.D., Wang, H., Pedersen, J.S., Horvath, L.G., Achen, M.G., Stacker, S.A., Risbridger, G.P., 2009. Elevated level of inhibin-[alpha] subunit is pro-tumourigenic and pro-metastatic and associated with extracapsular spread in advanced prostate cancer. Br. J. Cancer 100, 1784–1793. Bernabeu, C., Lopez-Novoa, J.M., Quintanilla, M., 2009. The emerging role of TGF[beta] superfamily coreceptors in cancer. Biochim. Biophys. Acta (BBA): Mol. Basis Dis. 1792, 954–973. Bernard, D.J., 2003. Editorial commentary: SMAD expression in the testis predicts age- and cell-specific responses to activin and TGFbeta. J. Androl. 24, 201–203. Bierie, B., Moses, H.L., 2010. TGF-b and cancer. Cytokine Growth Factor Rev. 17, 29– 40. Bilandzic, M., Chu, S., Farnworth, P.G., Harrison, C., Nicholls, P., Wang, Y., Escalona, R.M., Fuller, P.J., Findlay, J.K., Stenvers, K.L., 2009. Loss of betaglycan contributes to the malignant properties of human granulosa tumor cells. Mol. Endocrinol. 23, 539–548. Blair, C.R., Stone, J.B., Wells, R.G., 2011. The type III TGF-beta receptor betaglycan transmembrane-cytoplasmic domain fragment is stable after ectodomain cleavage and is a substrate of the intramembrane protease gamma-secretase. Biochim. Biophys. Acta 1813, 332–339. Blobe, G.C., Liu, X., Fang, S.J., How, T., Lodish, H.F., 2001. A novel mechanism for regulating transforming growth factor b (TGF-b) signaling. J. Biol. Chem. 276, 39608–39617. Boggess, J., Soules, M., Goff, B., Greer, B., Cain, J., Tamimi, H., 1997. Serum inhibin and disease status in women with ovarian granulosa cell tumors. Gynecol. Oncol. 64, 64–69. Brown, C.B., Boyer, A.S., Runyan, R.B., Barnett, J.V., 1999. Requirement of type III TGF-b receptor for endocardial cell transformation in the heart. Science 283, 2080–2082. Chand, A.L., Ooi, G.T., Harrison, C.A., Shelling, A.N., Robertson, D.M., 2007a. Functional analysis of the human inhibin a subunit variant A257T and its potential role in premature ovarian failure. Hum. Reprod. 22, 3241–3248. Chand, A.L., Robertson, D.M., Shelling, A.N., Harrison, C.A., 2007b. Mutational analysis of betaglycan/TGF-[beta]RIII in premature ovarian failure. Fertil. Steril. 87, 210–212. Cheifetz, S., Andres, J.L., Massague, J., 1988. The transforming growth factor-b receptor type III is a membrane proteoglycan. J. Biol. Chem. 263, 16984–16991. Cheifetz, S., Hernandez, H., Laiho, M., ten Dijke, P., Iwata, K., Massagué, J., 1990. Distinct transforming growth factor-beta (TGF-beta) receptor subsets as determinants of cellular responsiveness to three TGF-beta isoforms. J. Biol. Chem. 265, 20533–20538. Chen, W., Kirkbride, K.C., How, T., Nelson, C.D., Mo, J., Frederick, J.P., Wang, X.-F., Lefkowitz, R.J., Blobe, G.C., 2003. b-Arrestin 2 mediates endocytosis of type III TGF-b receptor and down-regulation of its signaling. Science 301, 1394–1397. Chen, Y.-G., 2009. Endocytic regulation of TGF-beta signaling. Cell Res. 19, 58–70. Cheung, H., Mei, J., Xu, R., 2004. Quantification of soluble betaglycan in porcine milk. Asia Pac. J. Clin. Nutr., 12. Compton, L.A., Potash, D.A., Brown, C.B., Barnett, J.V., 2007. Coronary vessel development is dependent on the type III transforming growth factor {beta} receptor. Circ. Res. 101, 784–791. Cooper, S.J., Zou, H., LeGrand, S.N., Marlow, L.A., von Roemeling, C.A., Radisky, D.C., Wu, K.J., Hempel, N., Margulis, V., Tun, H.W., Blobe, G.C., Wood, C.G., Copland, J.A., 2010. Loss of type III transforming growth factor-[beta] receptor expression is due to methylation silencing of the transcription factor GATA3 in renal cell carcinoma. Oncogene 29, 2905–2915. Copland, J., Luxon, B., Ajani, L., Maity, T., Campagnaro, E., Guo, H., LeGrand, S., Tamboli, P., Wood, C., 2003. Genomic profiling identifies alterations in TGFbeta signaling through loss of TGFbeta receptor expression in human renal cell carcinogenesis and progression. Oncogene 22, 8053–8062. Criswell, T.L., Arteaga, C.L., 2007. Modulation of NF{kappa}B activity and E-cadherin by the type III transforming growth factor beta receptor regulates cell growth and motility. J. Biol. Chem. 282, 32491–32500. Criswell, T.L., Dumont, N., Barnett, J.V., Arteaga, C.L., 2008. Knockdown of the transforming growth factor-b type III receptor impairs motility and invasion of metastatic cancer cells. Cancer Res. 68, 7304–7312. de Fraipont, F., El Atifi, M., Cherradi, N., Le Moigne, G., Defaye, G., Houlgatte, R., Bertherat, J., Bertagna, X., Plouin, P.F., Baudin, E., Berger, F., Gicquel, C., Chabre, O., Feige, J.J., 2005. Gene expression profiling of human adrenocortical tumors using complementary deoxyribonucleic acid microarrays identifies several candidate genes as markers of malignancy. J. Clin. Endocrinol. Metab. 90, 1819– 1829. Denzer, K., Kleijmeer, M.J., Heijnen, H.F., Stoorvogel, W., Geuze, H.J., 2000. Exosome: from internal vesicle of the multivesicular body to intercellular signaling device. J. Cell Sci. 113, 3365–3374.

M. Bilandzic, K.L. Stenvers / Molecular and Cellular Endocrinology 359 (2012) 13–22 Dixit, H., Rao, K.L., Padmalatha, V.V., Kanakavalli, M., Deenadayal, M., Gupta, N., Chakrabarty, B.N., Singh, L., 2006. Mutational analysis of the betaglycan genecoding region in susceptibility for ovarian failure. Hum. Reprod. 21, 2041–2046. Dong, M., How, T., Kirkbride, K., Gordon, K., Lee, J., Hempel, N., Kelly, P., Moeller, B., Marks, J., Blobe, G., 2007. The type III TGF-beta receptor suppresses breast cancer progression. J. Clin. Invest. 117, 206–217. Drummond, A.E., Le, M.T., Ethier, J.-F., Dyson, M., Findlay, J.K., 2002. Expression and localization of activin receptors, Smads, and {beta}glycan to the postnatal rat ovary. Endocrinology 143, 1423–1433. Drummond, A.E., Mitzi, D., Le, M.T., Ethier, J.-F., Findlay, J.K., 2003. Ovarian follicle populations of the rat express TGF-[beta] signalling pathways. In: Proceedings of the Fifth Sapporo International Symposium on Ovarian Function: Basic and Clinical Updates in Ovarian Function, vol. 202, pp. 53–57. Eickelberg, O., Centrella, M., Reiss, M., Kashgarian, M., Wells, R.G., 2002. Betaglycan inhibits TGF-b signaling by preventing type I–type II receptor complex formation. J. Biol. Chem. 277, 823–829. Escalona, R.M., Stenvers, K.L., Farnworth, P.G., Findlay, J.K., Ooi, G.T., 2009. Reducing betaglycan expression by RNA interference (RNAi) attenuates inhibin bioactivity in L[beta]T2 gonadotropes. Mol. Cell. Endocrinol. 307, 149–156. Esparza-López, J., Montiel, J.L., Vilchis-Landeros, M.M., Okadome, T., Miyazono, K., López-Casillas, F., 2001. Ligand binding and functional properties of betaglycan, a co-receptor of the transforming growth factor-b superfamily. J. Biol. Chem. 276, 14588–14596. Ethier, J.-F., Farnworth, P.G., Findlay, J.K., Ooi, G.T., 2002. Transforming growth factor-{beta} modulates inhibin A bioactivity in the L{beta}T2 gonadotrope cell line by competing for binding to betaglycan. Mol. Endocrinol. 16, 2754–2763. Farnworth, P.G., Stanton, P.G., Wang, Y., Escalona, R., Findlay, J.K., Ooi, G.T., 2006a. Inhibins differentially antagonize activin and bone morphogenetic protein action in a mouse adrenocortical cell line. Endocrinology 147, 3462–3471. Farnworth, P.G., Wang, Y., Escalona, R., Leembruggen, P., Ooi, G.T., Findlay, J.K., 2007. Transforming growth factor-{beta} blocks inhibin binding to different target cell types in a context-dependent manner through dual mechanisms involving betaglycan. Endocrinology 148, 5355–5368. Farnworth, P.G., Wang, Y., Leembruggen, P., Ooi, G.T., Harrison, C., Robertson, D.M., Findlay, J.K., 2006b. Rodent adrenocortical cells display high affinity binding sites and proteins for inhibin A, and express components required for autocrine signalling by activins and bone morphogenetic proteins. J. Endocrinol. 188, 451–465. Finger, E.C., Lee, N.Y., You, H.-J., Blobe, G.C., 2008a. Endocytosis of the type III transforming growth factor-b (TGF-b) receptor through the clathrinindependent/lipid raft pathway regulates TGF-b signaling and receptor downregulation. J. Biol. Chem. 283, 34808–34818. Finger, E.C., Turley, R.S., Dong, M., How, T., Fields, T.A., Blobe, G.C., 2008b. TGFBRIII suppresses non-small cell lung cancer invasiveness and tumorigenicity. Carcinogenesis 29, 528–535. Florio, P., Ciarmela, P., Reis, F.M., Toti, P., Galleri, L., Santopietro, R., Tiso, E., Tosi, P., Petraglia, F., 2005. Inhibin {alpha}-subunit and the inhibin coreceptor betaglycan are downregulated in endometrial carcinoma. Eur. J. Endocrinol. 152, 277–284. Fuller, P.J., Chu, S., 2004. Signalling pathways in the molecular pathogenesis of ovarian granulosa cell tumours. Trends Endocrinol. Metab. 15, 122–128. Gatza, C.E., Oh, S.Y., Blobe, G.C., 2010. Roles for the type III TGF-[beta] receptor in human cancer. Cell. Signal. 22, 1163–1174. Glister, C., Satchell, L., Knight, P.G., 2010. Changes in expression of bone morphogenetic proteins (BMPs), their receptors and inhibin co-receptor betaglycan during bovine antral follicle development: inhibin can antagonize the suppressive effect of BMPs on thecal androgen production. Reproduction 140, 699–712. Gordon, K.J., Dong, M., Chislock, E.M., Fields, T.A., Blobe, G.C., 2008. Loss of type III transforming growth factor b receptor expression increases motility and invasiveness associated with epithelial to mesenchymal transition during pancreatic cancer progression. Carcinogenesis 29, 252–262. Gordon, K.J., Kirkbride, K.C., How, T., Blobe, G.C., 2009. Bone morphogenetic proteins induce pancreatic cancer cell invasiveness through a Smad1-dependent mechanism that involves matrix metalloproteinase-2. Carcinogenesis 30, 238–248. Hamar, B.D., Buhimschi, I.A., Sfakianaki, A.K., Pettker, C.M., Magloire, L.K., Funai, E.F., Copel, J.A., Buhimschi, C.S., 2006. Serum and urine inhibin A but not free activin A are endocrine biomarkers of severe pre-eclampsia. Am. J. Obstet. Gynecol. 195, 1636–1645. Hempel, N., How, T., Cooper, S.J., Green, T.R., Dong, M., Copland, J.A., Wood, C.G., Blobe, G.C., 2008. Expression of the type III TGF-b receptor is negatively regulated by TGF-b. Carcinogenesis 29, 905–912. Hempel, N., How, T., Dong, M., Murphy, S.K., Fields, T.A., Blobe, G.C., 2007. Loss of betaglycan expression in ovarian cancer: role in motility and invasion. Cancer Res. 67, 5231–5238. Hill, C.S., 2009. Nucleocytoplasmic shuttling of Smad proteins. Cell Res. 19, 36–46. Hofland, J., Timmerman, M.A., de Herder, W.W., van Schaik, R.H., de Krijger, R.R., de Jong, F.H., 2006. Expression of activin and inhibin subunits, receptors and binding proteins in human adrenocortical neoplasms. Clin. Endocrinol. (Oxf.) 65, 792–799. Hsueh, A.J., Dahl, K.D., Vaughan, J., Tucker, E., Rivier, J., Bardin, C.W., Vale, W., 1987. Heterodimers and homodimers of inhibin subunits have different paracrine action in the modulation of luteinizing hormone-stimulated androgen biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 84, 5082–5086.

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Ikushima, H., Miyazono, K., 2010. TGFb signalling: a complex web in cancer progression. Nat. Rev. Cancer 10, 415–424. Itman, C., Mendis, S., Barakat, B., Loveland, K.L., 2006. All in the family: TGF-beta family action in testis development. Reproduction 132, 233–246. Jullien, J., Gurdon, J., 2005. Morphogen gradient interpretation by a regulated trafficking step during ligand–receptor transduction. Genes Dev. 19, 2682– 2694. Kardassis, D., Murphy, C., Fotsis, T., Moustakas, A., Stournaras, C., 2009. Control of transforming growth factor b signal transduction by small GTPases. FEBS J. 276, 2947–2965. Kirkbride, K.C., Townsend, T.A., Bruinsma, M.W., Barnett, J.V., Blobe, G.C., 2008. Bone morphogenetic proteins signal through the transforming growth factor-{beta} type III receptor. J. Biol. Chem. 283, 7628–7637. Knight, P.G., Glister, C., 2006. TGF-{beta} superfamily members and ovarian follicle development. Reproduction 132, 191–206. Kovacs, J.J., Hara, M.R., Davenport, C.L., Kim, J., Lefkowitz, R.J., 2009. Arrestin development: emerging roles for [beta]-arrestins in developmental signaling pathways. Dev. Cell 17, 443–458. Kumanov, P., Nandipati, K., Tomova, A., Robeva, R., Agarwal, A., 2005. Significance of inhibin in reproductive pathophysiology and current clinical applications. Reprod. Biomed. 10, 786–812. Le Roy, C., Leduque, P., Yuan Li, J., Saez, J.M., Langlois, D., 1998. Antisense oligonucleotide targeting the transforming growth factor beta1 increases expression of specific genes and functions of Leydig cells. Eur. J. Biochem. 257, 506–514. Le Roy, C., Lejeune, H., Chuzel, F., Saez, J.M., Langlois, D., 1999. Autocrine regulation of Leydig cell differentiated functions by insulin-like growth factor I and transforming growth factor beta. J. Steroid Biochem. Mol. Biol. 69, 379–384. Lee, J.D., Hempel, N., Lee, N.Y., Blobe, G.C., 2010. The type III TGF-b receptor suppresses breast cancer progression through G IPC-mediated inhibition of TGF-b signaling. Carcinogenesis 31, 175–183. Lee, N.Y., Kirkbride, K.C., Sheu, R.D., Blobe, G.C., 2009. The transforming growth factor-{beta} type III receptor mediates distinct subcellular trafficking and downstream signaling of activin-like kinase (ALK)3 and ALK6 receptors. Mol. Biol. Cell 20, 4362–4370. Lewis, K., Gray, P., Blount, A., MacConell, L., Wiater, E., Bilezikjian, L., Vale, W., 2000. Betaglycan binds inhibin and can mediate functional antagonism of activin signalling. Nature 404, 411–414. Lin, T., Calkins, J.K., Morris, P.L., Vale, W., Bardin, C.W., 1989. Regulation of Leydig cell function in primary culture by inhibin and activin. Endocrinology 125, 2134–2140. Ling, N., Ying, S.-Y., Ueno, N., Shimasaki, S., Esch, F., Hotta, M., Guillemin, R., 1986. Pituitary FSH is released by a heterodimer of the [beta]-subunits from the two forms of inhibin. Nature 321, 779–782. Liu, J., Kuulasmaa, T., Kosma, V.-M., Butzow, R., Vanttinen, T., Hyden-Granskog, C., Voutilainen, R., 2003. Expression of betaglycan, an inhibin coreceptor, in normal human ovaries and ovarian sex cord-stromal tumors and its regulation in cultured human granulosa-luteal cells. J. Clin. Endocrinol. Metab. 88, 5002– 5008. Lönn, P., Morén, A., Raja, E., Dahl, M., Moustakas, A., 2009. Regulating the stability of TGFbold beta receptors and Smads. Cell Res. 19, 21–35. Looyenga, B.D., Wiater, E., Vale, W., Hammer, G.D., 2010. Inhibin-A antagonizes TGF{beta}2 signaling by down-regulating cell surface expression of the TGF{beta} coreceptor betaglycan. Mol. Endocrinol. 24, 608–620. López-Casillas, F., Cheifetz, S., Doody, J., Andres, J.L., Lane, W.S., Massague, J., 1991. Structure and expression of the membrane proteoglycan betaglycan, a component of the TGF-[beta] receptor system. Cell 67, 785–795. López-Casillas, F., Payne, H., Andres, J., Massague, J., 1994. Betaglycan can act as a dual modulator of TGF-beta access to signaling receptors: mapping of ligand binding and GAG attachment sites. J. Cell Biol. 124, 557–568. López-Casillas, F., Wrana, J.L., Massagué, J., 1993. Betaglycan presents ligand to the TGF[beta] signaling receptor. Cell 73, 1435–1444. Loveland, K.L., Dias, V., Meachem, S., Rajpert-De Meyts, E., 2007. The transforming growth factor-beta superfamily in early spermatogenesis: potential relevance to testicular dysgenesis. Int. J. Androl. 30, 377–384, discussion 384. Makanji, Y., Kl, W., Mc, W., Kl, C., Dm, R., Ca, H., 2008. Suppression of inhibin A biological activity by alterations in the binding site for betaglycan. J. Biol. Chem. 283, 16743–16751. Makanji, Y., Temple-Smith, P.D., Walton, K.L., Harrison, C.A., Robertson, D.M., 2009. Inhibin B is a more potent suppressor of rat follicle-stimulating hormone release than inhibin a in vitro and in vivo. Endocrinology 150, 4784–4793. Margulis, V., Maity, T., Zhang, X.-Y., Cooper, S.J., Copland, J.A., Wood, C.G., 2008. Type III transforming growth factor-b (TGF-b) receptor mediates apoptosis in renal cell carcinoma independent of the canonical TGF-b signaling pathway. Clin. Cancer Res. 14, 5722–5730. Matzuk, M., Finegold, M., Su, J., Hsueh, A., Bradley, A., 1992. Alpha-inhibin is a tumour-suppressor gene with gonadal specificity in mice. Nature 360, 313–319. Mauduit, C., Chauvin, M.A., de Peretti, E., Morera, A.M., Benahmed, M., 1991. Effect of activin A on dehydroepiandrosterone and testosterone secretion by primary immature porcine Leydig cells. Biol. Reprod. 45, 101–109. McLean, S., Di Guglielmo, G.M., 2010. TGFb(transforming growth factor b) receptor type III directs clathrin-mediated endocytosis of TGFb receptor types b I and II. Biochem. J. 429, 137–145. Mendoza, V., Vilchis-Landeros, M.M., Mendoza-Hernandez, G., Huang, T., Villarreal, M.M., Hinck, A.P., López-Casillas, F., Montiel, J.-L., 2009. Betaglycan has two independent domains required for high affinity TGFb binding: proteolytic

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cleavage separates the domains and inactivates the neutralizing activity of the soluble receptor. Biochemistry 48, 11755–11765. Mom, C., Engelen, M., Willemse, P., Gietema, J., ten Hoor, K., de Vries, E., van der Zee, A., 2007. Granulosa cell tumors of the ovary: the clinical value of serum inhibin A and B levels in a large single center cohort. Gynecol. Oncol. 105, 365–372. Mythreye, K., Blobe, G.C., 2009. The type III TGF-b receptor regulates epithelial and cancer cell migration through b-arrestin2-mediated activation of Cdc42. Proc. Natl. Acad. Sci. U.S.A. 106, 8221–8226. Olaso, R., Pairault, C., Saez, J.M., Habert, R., 1999. Transforming growth factor beta3 in the fetal and neonatal rat testis: immunolocalization and effect on fetal Leydig cell function. Histochem. Cell Biol. 112, 247–254. Omori, Y., Nakamura, K., Yamashita, S., Matsuda, H., Mizutani, T., Miyamoto, K., Minegishi, T., 2005. Effect of follicle-stimulating hormone and estrogen on the expression of betaglycan messenger ribonucleic acid levels in cultured rat granulosa cells. Endocrinology 146, 3379–3386. Pepin, M.C., Beauchemin, M., Plamondon, J., O’Connor-McCourt, M.D., 1994. Mapping of the ligand binding domain of the transforming growth factor beta receptor type III by deletion mutagenesis. Proc. Natl. Acad. Sci. U.S.A. 91, 6997– 7001. Risbridger, G.P., Clements, J., Robertson, D.M., Drummond, A.E., Muir, J., Burger, H.G., de Kretser, D.M., 1989. Immuno- and bioactive inhibin and inhibin alphasubunit expression in rat Leydig cell cultures. Mol. Cell. Endocrinol. 66, 119– 122. Robertson, D.M., Oehler, M.K., 2005. Emerging role of inhibin as a biomarker for ovarian cancer. Women’s Health 1, 51–57. Sarraj, M.A., Chua, H.K., Umbers, A., Loveland, K.L., Findlay, J.K., Stenvers, K.L., 2007. Differential expression of TGFBR3 (betaglycan) in mouse ovary and testis during gonadogenesis. Growth Factors 25, 334–345. Sarraj, M.A., Escalona, R.M., Umbers, A., Chua, H.K., Small, C., Griswold, M., Loveland, K., Findlay, J.K., Stenvers, K.L., 2010. Fetal testis dysgenesis and compromised leydig cell function in Tgfbr3 (Betaglycan) knockout mice. Biol. Reprod. 82, 153– 162. Segarini, P.R., Rosen, D.M., Seyedin, S.M., 1989. Binding of transforming growth factor-{beta} to cell surface proteins varies with cell type. Mol. Endocrinol. 3, 261–272. Sharifi, N., Hurt, E.M., Kawasaki, B.T., Farrar, W.L., 2007. TGFBR3 loss and consequences in prostate cancer. Prostate 67, 301–311. Steller, M.D., Shaw, T.J., Vanderhyden, B.C., Ethier, J.-F., 2005. Inhibin resistance is associated with aggressive tumorigenicity of ovarian cancer cells. Mol. Cancer Res. 3, 50–61. Stenvers, K.L., Findlay, J.K., 2010. Inhibins: from reproductive hormones to tumor suppressors. Trends Endocrinol. Metab. 21, 174–180. Stenvers, K.L., Tursky, M.L., Harder, K.W., Kountouri, N., Amatayakul-Chantler, S., Grail, D., Small, C., Weinberg, R.A., Sizeland, A.M., Zhu, H.-J., 2003. Heart and liver defects and reduced transforming growth factor {beta}2 sensitivity in transforming growth factor {beta} type III receptor-deficient embryos. Mol. Cell. Biol. 23, 4371–4385. Tsigkou, A., Luisi, S., De Leo, V., Patton, L., Gambineri, A., Reis, F.M., Pasquali, R., Petraglia, F., 2008a. High serum concentration of total inhibin in polycystic ovary syndrome. Fertil. Steril. 90, 1859–1863. Tsigkou, A., Luisi, S., Reis, F.M., Petraglia, F., 2008b. Inhibins as diagnostic markers in human reproduction. Adv. Clin. Chem. 45, 1–29.

Tsigkou, A., Marrelli, D., Reis, F.M., Luisi, S., Silva-Filho, A.L., Roviello, F., Triginelli, S.A., Petraglia, F., 2007. Total inhibin is a potential serum marker for epithelial ovarian cancer. J. Clin. Endocrinol. Metab. 92, 2526–2531. Turley, R.S., Finger, E.C., Hempel, N., How, T., Fields, T.A., Blobe, G.C., 2007. The type III transforming growth factor-{beta} receptor as a novel tumor suppressor gene in prostate cancer. Cancer Res. 67, 1090–1098. Vale, W., Rivier, J., Vaughan, J., McClintock, R., Corrigan, A., Woo, W., Karr, D., Spiess, J., 1986. Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature 321, 776–779. Vanttinen, T., Kuulasmaa, T., Liu, J., Voutilainen, R., 2002. Expression of activin/ inhibin receptor and binding protein genes and regulation of activin/inhibin peptide secretion in human adrenocortical cells. J. Clin. Endocrinol. Metab. 87, 4257–4263. Vanttinen, T., Liu, J., Kuulasmaa, T., Kivinen, P., Voutilainen, R., 2003. Expression of activin/inhibin signaling components in the human adrenal gland and the effects of activins and inhibins on adrenocortical steroidogenesis and apoptosis. J. Endocrinol. 178, 479–489. Velasco-Loyden, G., Arribas, J., López-Casillas, F., 2004. The shedding of betaglycan is regulated by pervanadate and mediated by membrane type matrix metalloprotease-1. J. Biol. Chem. 279, 7721–7733. Walker, K.A., Sims-Lucas, S., Caruana, G., Cullen-McEwen, L., Li, J., Sarraj, M.A., Bertram, J.F., Stenvers, K.L., 2011. Betaglycan is required for the establishment of nephron endowment in the mouse. PLoS ONE 6 (4), e18723. http://dx.doi.org/ 10.1371/journal.pone.0018723. Wang, X.-F., Lin, H.Y., Ng-Eaton, E., Downward, J., Lodish, H.F., Weinberg, R.A., 1991. Expression cloning and characterization of the TGF-[beta] type III receptor. Cell 67, 797–805. Webber, J., Steadman, R., Mason, M.D., Tabi, Z., Clayton, A., 2010. Cancer exosomes trigger fibroblast to myofibroblast differentiation. Cancer Res. 70, 9621–9630. Wiater, E., Harrison, C.A., Lewis, K.A., Gray, P.C., Vale, W.W., 2006. Identification of distinct inhibin and transforming growth factor beta-binding sites on betaglycan: functional separation of betaglycan co-receptor actions. J. Biol. Chem. 281, 17011–17022. Wiater, E., Lewis, K.A., Donaldson, C., Vaughan, J., Bilezikjian, L., Vale, W., 2009. Endogenous betaglycan is essential for high-potency inhibin antagonism in gonadotropes. Mol. Endocrinol. 23, 1033–1042. Wiater, E., Vale, W., 2003. Inhibin is an antagonist of bone morphogenetic protein signaling. J. Biol. Chem. 278, 7934–7941. Wu, D.T., Bitzer, M., Ju, W., Mundel, P., Bottinger, E.P., 2005. TGF-b concentration specifies differential signaling profiles of growth arrest/differentiation and apoptosis in podocytes. J. Am. Soc. Nephrol. 16, 3211–3221. You, H.J., Bruinsma, M.W., How, T., Ostrander, J.H., Blobe, G.C., 2007. The type III TGF-b receptor signals through both Smad3 and the p38 MAP kinase pathways to contribute to inhibition of cell proliferation. Carcinogenesis 28, 2491–2500. You, H.J., How, T., Blobe, G.C., 2009. The type III transforming growth factor-b receptor negatively regulates nuclear factor kappa B signaling through its interaction with b-arrestin2. Carcinogenesis 30, 1281–1287. Zhu, R., Zhou, X., Chen, Y., Qiu, C., Xu, W., Shen, Z., 2009. Aberrantly increased mRNA expression of betaglycan, an inhibin co-receptor in the ovarian tissues in women with polycystic ovary syndrome. J. Obstet. Gynaecol. Res. 36, 138–146.