The activins and their binding protein, follistatin—Diagnostic and therapeutic targets in inflammatory disease and fibrosis

Cytokine & Growth Factor Reviews 24 (2013) 285–295

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The activins and their binding protein, follistatin—Diagnostic and therapeutic targets in inflammatory disease and fibrosis M.P. Hedger *, D.M. de Kretser 1 Monash Institute of Medical Research, Monash University, Melbourne, Victoria, Australia

A R T I C L E I N F O

A B S T R A C T

Article history: Available online 29 March 2013

The activins, as members of the transforming growth factor-b superfamily, are pleiotrophic regulators of cell development and function, including cells of the myeloid and lymphoid lineages. Clinical and animal studies have shown that activin levels increase in both acute and chronic inflammation, and are frequently indicators of disease severity. Moreover, inhibition of activin action can reduce inflammation, damage, fibrosis and morbidity/mortality in various disease models. Consequently, activin A and, more recently, activin B are emerging as important diagnostic tools and therapeutic targets in inflammatory and fibrotic diseases. Activin antagonists such as follistatin, an endogenous activin-binding protein, offer considerable promise as therapies in conditions as diverse as sepsis, liver fibrosis, acute lung injury, asthma, wound healing and ischaemia–reperfusion injury. Crown Copyright ß 2013 Published by Elsevier Ltd. All rights reserved.

Keywords: Activin Follistatin Inflammation Fibrosis Immunoregulation

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biology of the activins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. The activin family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Activin formation and origins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Regulation of activin production . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Activin receptors and signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Activin actions on cells and tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of macrophages and inflammatory responses . . . . . . . . . 3.1. Regulation of fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. 3.3. Immunoregulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endogenous regulation of activin activity. . . . . . . . . . . . . . . . . . . . . . . . . . . Follistatin and other activin-binding proteins . . . . . . . . . . . . . . . . . . 4.1. Inhibins and inhibitors of receptor interactions . . . . . . . . . . . . . . . . 4.2. Activin in inflammation, immunity and fibrosis. . . . . . . . . . . . . . . . . . . . . . Activin responses to inflammation in animal models and humans . 5.1. 5.2. Studies using the lipopolysaccharide-induced inflammation model Other inflammatory models and blocking studies . . . . . . . . . . . . . . 5.3. Potential for therapeutic and diagnostics applications in disease . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author at: Centre for Reproduction and Development, Monash Institute of Medical Research, Monash University, Clayton 3168, Victoria, Australia. Tel.: +61 3 9902 4758; fax: +61 3 9594 7114. E-mail addresses: [email protected] (M.P. Hedger), [email protected] (D.M. de Kretser). 1 Centre for Reproduction and Development, Monash Institute of Medical Research, Monash University, Clayton 3168, Victoria, Australia. Tel.: +61 3 9902 4730; fax: +61 3 9594 7114. 1359-6101/$ – see front matter . Crown Copyright ß 2013 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cytogfr.2013.03.003

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1. Introduction The activins exert a broad range of regulatory effects upon haematopoiesis, inflammation and immunity. Follistatin is an endogenous high-affinity activin-binding protein that can modulate the bioactivity of the activins. Overwhelming evidence of a fundamental role for the activins and follistatin in inflammation and fibrosis highlights the emerging potential for novel diagnostics and therapies that target these molecular regulators. This brief survey is intended to provide an outline the current state of knowledge surrounding the inflammatory and immunoregulatory properties of the activins and follistatin and identifies several potential applications involving these proteins in the therapy of inflammatory and fibrotic diseases. 2. Biology of the activins 2.1. The activin family The activins are a homologous family of homodimers and heterodimers belonging to the transforming growth factor-b (TGFb) superfamily of growth and differentiation cytokines. They were initially named for their roles in the reproductive system, specifically their ability to activate the release of the gonadotrophic hormone, follicle-stimulating hormone (FSH) from the anterior pituitary [1]. However, they were soon found to exert a number of effects on a range of systems, including haematopoiesis and immune cell development [2–4]. Currently, there is a considerable degree of interest in the steady stream of new evidence for their fundamental roles in inflammation and immunity (see [5] for comprehensive review). In this role, the activins share similarities with the TGFbs themselves, which have long been recognised as pleiotrophic immunoregulators. However, the activins possess many unique properties distinct from the TGFbs, including their own unique receptors and complex mechanisms of activity regulation. The first-identified and best studied family member is activin A, initially isolated from the ovary, and subsequently from cultures of cells of bone marrow origin (leukaemia cells and bone marrow stromal cells) [6,7]. This protein is a homodimer of the bA subunit of the gonadal hormone, inhibin A, which is a heterodimer of a and b subunits. Activin B is a homodimer of the bB subunit of inhibin B, which has about 65% sequence homology with the bA subunit [8]. Heterodimers of the two b subunits, comprising activin AB, have also been identified. Most studies on the functional properties of the activins to date have focussed on the activin A homodimer, but there is increasing evidence for the involvement of activin B, as well [9]. Both activin A and B are widely expressed. Several other activin subunits have been discovered, including bC and bE, which appear to be mostly, although not entirely, confined to the liver [10,11]. Biological roles for activin C and E are only just beginning emerge [12–14]. In this review, the use of the terms ‘‘activin’’ and ‘‘activins’’ refers specifically to activin A or B, or both together. 2.2. Activin formation and origins The subunits of activin A and B are the products of two separate genes, named inhibin beta A (INHBA) and inhibin beta B (INHBB), respectively, because of their original identification as subunits of the gonadal hormone, inhibin [8,15]. Similar to the TGFbs, the activins are disulphide-linked dimers of approximately 25 kDa in mass, with intra-strand disulphide bonds that form a cysteine knot folding motif [16]. The activins A and B are highly conserved across species, displaying greater than 97% conservation between human and predicted marsupial, monotreme and bird sequences (data from NCBI database). This implies a high degree of individual

functional importance, particularly in contrast with the substantially lower sequence homology of approximately 65% between the two activin forms. They are synthesised as inactive disulphidelinked precursors, which are processed to the mature cytokine by enzymatic or acid hydrolysis (Fig. 1) [17,18]. In contrast to most other members of the TGFb superfamily, which circulate as latent precursor complexes, the activin propeptide sequence has a relatively weak affinity for the mature dimer, is easily displaced, and does not interfere with receptor binding [19,20]. Both activin A and B are readily measurable in the blood and other biological fluids using two-site ELISAs [21,22]. The bA and bB subunit genes are expressed in most, if not all, tissues. There appear to be significant species-specific differences in baseline tissue expression levels, but both genes are highly expressed in the male and female reproductive tracts under normal conditions, with high levels of bA subunit mRNA also reported in the bone marrow, central nervous system, placenta, liver, heart, adrenal glands and fat in different studies [23–25]. The bB subunit mRNA is likewise highly expressed in the central nervous system, but also in the placenta and salivary glands. It is unclear, however, how much of this subunit mRNA leads to production of bioactive activin homodimers in the various tissues, particularly as there seems to be a relatively poor relationship between mRNA expression levels and protein content for activin A in the mouse [25,26]. In the mouse, it is the bone marrow that contains the highest concentration of activin A protein, while mRNA expression in this tissue is relatively low [25]. At the cellular level, activin A is produced by myeloid-lineage cells (macrophages and dendritic cells), bone marrow stromal cells, epithelial cells, mast cells and endothelial cells, in particular [27–32]. In contrast to activin A, the cellular origins of activin B (and activin AB) have received very little attention, with many questions remaining about the principal sources and regulation of this protein. 2.3. Regulation of activin production The current state of understanding of the regulation of activin A has come from studies on monocyte/macrophages, placental cells, bone marrow stromal cells and epithelial cells of the testis and ovary, such as the Sertoli cells and granulosa cells [5]. These studies have established that activin A gene expression is stimulated via several pro-inflammatory and immunoregulatory signalling pathways. The bA subunit gene promoter comprises one or more sites that recognise the stress/inflammatory JUN/FOS transcription factor, AP-1, although there do not appear to be any typical consensus NF-kB sites within the proximal promoter [33–36]. Expression of the bA subunit is stimulated in a synergistic manner by the Th2 cell transcription factor c-MAF and NF-AT, a transcription factor that is expressed mainly in immune cells [37]. The presence of multiple phorbol ester-responsive elements (i.e. AP-1 and AP-2) in the promoter, and the stimulation of activin A production by phorbol esters in several cell types, also implicate protein kinase C in its regulation [33,38,39]. Consequently, activin A production is potently stimulated by inflammatory mediators that activate these pathways, including interleukin-1 (IL1), Tolllike receptor (TLR) ligands such as lipopolysaccharide (LPS), and tumour necrosis factor-a (TNFa), acting via MyD88/TRAF signalling and the MAP kinases [27,31,39–42]. While regulation of activin A at the level of gene expression is relatively well-defined, the control of subunit peptide synthesis, dimer formation and eventual secretion of the mature protein is not. Following translation into protein, activin A is stored by certain cell types, including various bone marrow cells and epithelial cells in the male reproductive tract [25,26], and release of activin A from the cell is clearly an important level of control of its activity [43]. Cell-specific differences in storage and release may

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Fig. 1. Activin production, signalling and sites of regulation. Activin A is a homodimer of inhibin bA subunits encoded by the INHBA gene. Binding of activin A causes oligomerisation of activin type 2 (ActR2) and type 1 (ActR1) receptors on the surface of target cells, initiating intracellular signalling via SMAD2/3 and MAP kinases, and inducing cellular differentiation, proliferation or apoptosis. Activin B (not shown here) acts in a similar manner. Regulation of activin activity can occur through concurrent production of the inhibin a-subunit and bC subunit, which form non-activating heterodimers (inhibin, activin AC and activin C) that compete for the activin receptor complex. Activin A activity is also regulated by the activin-binding protein, follistatin, and by serum carrier proteins, such as a2-macroglobulin. Regulation at the level of the receptor also involves the membrane-bound co-receptors, TGFBR3 and BAMBI. Full details are provided in the text.

be part of the explanation why there is an obvious discrepancy between mRNA and protein levels in various tissues in the mouse, for example the liver (high mRNA expression, low protein content) and the bone marrow (low mRNA expression, high protein content). However, most features of the regulation of this stored activin A and eventual release, in particular the regulatory agents responsible, remain to be clarified. The promoter of the bB subunit that forms activin B has been investigated in the rat and bovine, and multiple AP-1 and AP-2 sites have been identified, suggesting that activin B may be, at least in part, regulated in a similar manner to that of activin A [38,44,45]. Nonetheless, relatively little is known about the regulation of activin B production by inflammatory mediators. Expression of the bB subunit in the liver of the mouse is stimulated by systemic administration of LPS, implicating TLR signalling in its regulation [42].

subunits [9,50]. It is generally assumed that activin B is a relatively weaker agonist than activin A due to a lower affinity for the activin receptor complexes, but activin B may have a broader range of actions due to the ability to utilise ALK7 as well as ALK4. Binding of activin and oligomerisation of the receptor complex activates receptor serine/threonine kinase activity, leading to phosphorylation of the intracellular SMAD proteins 2 and 3, which then form a heteromeric transcription factor with SMAD4 [51]. This is the same transcription factor cascade activated by TGFb binding to its own receptor complexes, and SMAD2/3 signalling thus forms a common pathway for regulation of inflammation and immunity. Regulation of this signalling pathway occurs through the actions of the inhibitory SMAD proteins, SMAD6 and SMAD7 [51]. In addition, several alternative signalling pathways may be activated by activin, most notably inflammation and stressmediated pathways via TRAF6 and downstream activation of the MAP kinases, p38 MAPK, JNK and ERK1/2 [47,52–54].

2.4. Activin receptors and signalling 3. Activin actions on cells and tissues The activin receptors are similar to those of other members of the TGFb superfamily. Activins bind to one of two specific type 2 activin receptors (ACVR2A or ACVR2B) on the cell surface, which dimerise with an type 1 activin receptor serine/threonine kinase (activin receptor-like kinase, ALK) (Fig. 1) [46,47]. In the case of activin A, the type 1 receptor is ALK4 (ACVR1B), but activin B and activin AB can utilise either ALK4 or another type 1 receptor, ALK7 (ACVR1C) [48,49]. Observed differences in activin A and B activity may be attributed to different affinities for the type 1 and 2 receptors, and different cell and tissue distributions of the receptor

The activins exert a broad range of actions on a broad range of cells types and tissues, thereby regulating processes as diverse as gonadotrophin synthesis by the anterior pituitary, induction of the mesoderm during development, neurogenesis, apoptosis of liver cells and tumour angiogenesis [1,55–58]. In terms of effects on the immune system, among the earliest activin actions identified were the ability to regulate the development and activity of haematopoietic cells and immune cells from the thymus and bone marrow [3,4].

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3.1. Regulation of macrophages and inflammatory responses Studies using primary cultures and cell lines of the monocyte/ macrophage lineage have shown that activin A can induce IkB degradation and translocation of NF-kB into the nucleus, activate p38 MAPK and ERK1/2 signalling and stimulate the production of inflammatory mediators, including IL1b, TNFa, IL6, nitric oxide and prostanoids [59–62]. However, the role of activin A in regulating these cells is actually more sophisticated: in activated macrophages, the effects of activin A on inflammatory signalling and production of mediators tend to be inhibitory. Activin A inhibited processing of the IL1b precursor into the mature cytokine and increased production of the IL1 receptor antagonist (IL1ra) in human monocyte cell lines activated by LPS and phorbol esters [63]. Likewise, in various rat, mouse and human monocyte/ macrophage cultures, activin A inhibited the LPS-induced production of a broad range of key pro-inflammatory mediators, including IL1b, TNFa, IL6, IL8 and inducible nitric oxide synthase (iNOS), as well as the LPS-receptor, TLR4 and its co-receptor, CD14 [64–70]. Furthermore, activin A inhibits many of the actions mediated by IL1 and IL6 [6,71–73]. These various studies indicate that activin A, particularly when present at lower concentrations, promotes inflammation mediated by resting monocyte/macrophages, but opposes the inflammatory activity of these cells once inflammation is established and activin A concentrations increase. A similar role for activin B can be anticipated, but remains to be explored.

[30,40,91,95], and activin A is produced by activated murine CD4+ Th2 cells [37]. This observation, together with the fact that activin A has also been implicated in promoting Th2-regulated immune responses, such as asthma and atopy [77,86,96–100], mast cell recruitment, maturation and activity [32,80], and immunoglobulin production by B cells [59,101], has led to suggestions that activin A may be a Th2 cytokine. Studies have likewise attempted to demonstrate that activin A regulates macrophage polarisation, switching them between the classic pro-inflammatory M1 and alternatively activated, anti-inflammatory M2 subsets [37,68,102,103]; however, the conflicting conclusions reached by researchers in different studies highlights the fact that the role of activin A in macrophage development is actually determined by the doses employed, prior priming of the cells and, just possibly, the selective choice of functional readouts. Consistent with this immunoregulatory role, activin A also directly or indirectly inhibits thymocyte and peripheral T cell activation and growth, possibly through regulation of the production and activity of IL1, IL6 and IFNg [4,71,104], and induces apoptosis of both normal and transformed B cells [6,105,106]. Putting all the data together, it may not be sensible to define activin A as a Th1 or Th2 segregated cytokine, because it plays multiple, fundamental roles in both type 1 and type 2 immune responses. What is very clear, nonetheless, is that activin A is a significant regulator of the immune response, implicated in maintaining ‘‘immunosuppression’’ and tolerance, as well directing inflammation and fibrosis (Fig. 2).

3.2. Regulation of fibrosis 4. Endogenous regulation of activin activity Activin A stimulates fibroblast proliferation and differentiation into myofibroblasts, which are essential steps in the process of fibrosis following inflammation or trauma [4,74–76]. Furthermore, activin A stimulates renal and lung fibroblasts and pancreatic stellate cells to produce TGFb1, which is a central regulator of the fibrotic response [77,78]. Activin A is also able to induce the expression of TGFb1 and other regulators and intermediates of fibrosis, such as connective tissue growth factor, tissue inhibitor of metalloproteinase-1, plasminogen activator inhibitor 1, endothelin and type 1 collagen, in a range of cell types [76,79–82]. Moreover, TGFb itself has been shown to stimulate production of activin A itself by fibroblasts and synoviocytes, and expression of the bB subunit in ovarian cells [76,77,83–85]. Other regulators of fibrosis, such as TNFa, IL13, endothelin, angiotensin and thrombin also drive activin A production in various cell types and tissues [39,40,86–88]. These data implicate activin A, and potentially activin B as well, as a fundamental regulator and an important intermediary of the fibrotic process. 3.3. Immunoregulation In addition to regulation of monocyte/macrophage activation and function during inflammation, activin A exerts other critical immunoregulatory actions, which should briefly be mentioned. Activin A stimulates the recruitment and development of monocyte-derived dendritic cells [89–91], but inhibits the ability of dendritic cells to mature and stimulate T cell activation [30,92,93]. This inhibition is associated with a switch in production by NK cells from interferon-g (IFNg), a crucial stimulator of dendritic cell maturation, to the anti-inflammatory, immunosuppressive cytokine, IL-10 [93]. Similar inhibitory effects of activin A on the antigen-presenting activity of macrophages have been observed [70]. Activin A can also stimulate development of antigen-specific CD4+CD25+Foxp3+ regulatory T cells (Treg cells) [94]. Dendritic cells (and macrophages) produce activin A when activated by LPS and other TLR ligands or CD40L, a dendritic cell coreceptor ligand expressed on the surface of activated T cells

4.1. Follistatin and other activin-binding proteins Follistatin is an activin-binding protein that is widely expressed throughout the body, like the activins, but is not a member of the TGFb superfamily. Although the product of a single gene, alternative splicing produces two mRNA transcripts, which encode two mature proteins of 288 amino acids (FST288) and 315 amino acids (FST315), and glycosylation produces several additional variants ranging in size from 31 to 42 kDa [107–109]. The molecule contains an activin-binding site, and the affinity of follistatin for the activins is comparable to the affinity of the activins for the activin receptors (Fig. 3) [108,110,111]. Follistatin also possesses a heparin-binding sequence, which allows FST288 to bind intrinsically to heparan-sulphate proteoglycans on cell surfaces [109,112]. The C-terminal extension of FST315 normally obstructs the heparin-binding site, which allows the protein to circulate, but the site becomes exposed after binding to activin. Both proteins act to clear activin from the circulation by attachment to the cell surface and removal by a lysosomal degradation pathway [113]. Consequently, follistatin is an important biological regulator of activin activity in vivo, and this is believed to be its principal biological activity. However, follistatin also binds with lower affinity to several other members of the TGFb superfamily, including growth and differentiation factor-9 (GDF9), myostatin (GDF8), and several of the bone morphogenetic proteins (BMPs) [114–116]. Follistatin does not bind to TGFb1 or 2, but binding to TGFb3 has been reported [117]. Some of the observed biological effects of follistatin, such as the ability to stimulate muscle growth through inhibition of myostatin [114], can be attributed to these lower-affinity binding interactions. The biological activity of activin is regulated by a number of other molecules with activin-binding activity. Follistatin-like 3 (FSTL3, also called follistatin-related protein) is structurally related to follistatin and binds the activins with high affinity, but lacks the heparin-binding sequence of follistatin, and is considerably less effective at blocking endogenous activin activity [110,118]. The

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Fig. 2. Cellular sites of activin A production and actions in the immune system. Activin A is produced primarily by cells of myeloid origin (monocytes, macrophages, dendritic cells, neutrophils and mast cells), although production by Th2 lymphocytes also has been observed (red arrows). Activin regulates the function of most, if not all, immune cell types, but direct effects on the neutrophils, eosinophils and most T cell subsets (including the Th1 and Th2 cell subsets: broken arrows) remain to be established.

Fig. 3. Schematic diagram of the interaction between activin and follistatin. Both follistatin variants (FST288 and FST315) are produced from a single gene by alternate splicing and comprise an N-terminal domain and three cysteine-rich FS domains (FS1–3). The activin-binding region is located within the N-terminal region and encompasses the first two FS domains. The follistatins binds to the activin dimer in a ratio of 2:1. There is a heparin-binding motif within the FS1 domain, which allows follistatin to bind to both heparin and heparan sulphate-containing proteoglycans on cell surfaces. FS315 has a c-terminal extension that obstructs the heparin binding motif (broken arrow) prior to activin binding.

activins also bind reversibly to several other proteins in biological fluids, such as a2-macroglobulin [119]. As mentioned above, the activin propeptide sequence remains bound to the mature protein after secretion, although with relatively low affinity [19,20]. The propeptide sequence and molecules like a2-macroglobulin are not able to prevent activin receptor binding, but they may act as carriers for activin in biological fluids, possibly modulating the rate of clearance and, hence, effective bioactivity. 4.2. Inhibins and inhibitors of receptor interactions Several molecules act to interfere with the ability of the activins to activate their receptors. The inhibins, A and B, are able to bind to

the type 2 activin receptor subunit through the mediation of a coreceptor called TGFb receptor 3 (TGFBR3), or betaglycan, but they prevent the dimerisation of the type 2 and type 1 receptor that is necessary to initiate intracellular signalling [50,120]. The inhibins are chiefly produced by the gonads and circulate as hormone in the blood – they suppress activin activity at its sites of action, for example, inhibiting the release of FSH by the anterior pituitary [121]. At sites of production, however, the inhibin a subunit effectively competes with the b subunits to reduce formation of activin dimers [1,122]. As a result, the a subunit and the inhibins are highly specific regulators of activin action. There is also evidence that the activin bC subunit plays a similar role to that of the a subunit. Activin C and AC dimers lack the ability to activate

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activin receptor signalling [123,124], but overexpression of the bC subunit in mice was shown to cause inflammatory lesions in the liver, where the subunit is normally most highly expressed, along with reduced nuclear translocation of SMAD2 [12]. As it is unlikely that activin C itself is pro-inflammatory, these data suggest that the bC subunit can interfere with anti-inflammatory or immunoregulatory actions of activin by dimerising with the bA subunit and reducing production of activin A dimers, or though the ability of activin AC or C dimers to competitively inhibit activin binding to its receptor [12,123]. There is no evidence that overexpression of the bE subunit causes similar pathologies [14]. In summary, multiple homologous inhibin/activin subunits and dimers formed from these subunits can act as both paracrine and endocrine antagonists of activin bioactivity, including control of inflammation and immunoregulation (Fig. 1). At a more subtle level, competition between activin B and activin A may also be an important modulator of activin bioactivity, due to their differences in receptor affinities. In addition, several membrane-associated proteins interfere with the interaction between activin and its receptors. These include the bone morphogenetic protein and activin membranebound inhibitor (BAMBI), which is a transmembrane protein with homology to the activin type 1 receptors, but which lacks the intracellular kinase domain required for signalling, endoglin/ CD105 and the GPI-linked membrane protein, Cripto [125–127]. The resulting complexity of activin regulation is summarised and highlighted in Fig. 1. It is very obvious that the activins are kept under control at multiple levels, presumably because of the potentially deleterious effects of unregulated activin action. 5. Activin in inflammation, immunity and fibrosis 5.1. Activin responses to inflammation in animal models and humans As outlined in the previous section, activin A and B regulate the activity of many cell types involved in inflammation, immunity and fibrosis. Moreover, the activins (and, frequently, follistatin) are elevated in the blood and in the epithelial/endothelial, stromal and myeloid/lymphoid cells of affected tissues in numerous clinical and animal models of acute and chronic inflammation, fibrosis and cachexia [5], and there is increasing evidence for a direct relationship between activin A levels and the severity of the illness [81,128,129]. These associations suggest important roles for the activins in inflammatory and immunological disease, but what, exactly, are those roles?

activity, although it was reduced by cycloheximide, suggesting that new protein synthesis was involved [25]. Tissues with high endogenous levels of bA subunit mRNA, such as the liver in the case of the mouse, may have been responsible for this new synthesis. Measurement of activin A protein levels in a range of tissues before and after LPS-treatment indicated that the initial peak was also attributable to the release of stored activin A, particularly from the bone marrow, which displayed the highest concentration of preexisting activin A under normal conditions [25]. These findings provide an explanation for the very rapid increase in activin A levels in the blood in the acute LPS-induced inflammation model. By contrast, the regulation of activin B following LPS-treatment appears to occur primarily at the level of mRNA transcription (data not published). In the murine bone marrow, the majority of pre-stored activin A was immunolocalised to the neutrophil precursor population, and the decline in bone marrow activin A concentrations 1 h after LPStreatment was matched by a reduction in these activin Acontaining cells in the tissue sections [25]. This decline in the bone marrow was mirrored by an increase in activin A-containing neutrophils in the lungs, which was the only tissue apart from the blood that displayed an increase in activin A protein content after LPS-treatment. Isolated murine and human neutrophils spontaneously release their activin A in culture and this release is stimulated by TNFa, although not by LPS itself [132,133]. This release does not appear to be due to neutrophil degranulation or autolysis, but probably involves an active secretion process, yet to be elucidated. These data identify the neutrophils as an important, but previously under-investigated, source of activin A during acute inflammation, responsible for the rapid elevation of activin A in the blood and in tissues, such as the lung, where neutrophils reside after their release from the bone marrow (Fig. 4). It is important to note that the initial peak of activin A release following LPS administration is not accompanied by a contemporaneous increase in serum follistatin levels, indicating that this activin A can exert its effects unopposed. However, a broad increase in serum follistatin occurs following the first activin A peak. This increase in follistatin lasts for 4–10 h and peaks at about 6 h after treatment, corresponding with elevated hepatic follistatin mRNA expression [25]. The fact that this increased secretion of follistatin does not occur in mice given an excess of exogenous follistatin (1 mg, i.p.) 1 h prior to LPS administration, in order to

5.2. Studies using the lipopolysaccharide-induced inflammation model The most detailed experimental examination of role of activin in inflammation has employed a systemic LPS-induced inflammation model. In studies with mice and sheep, activin A in the blood was found to increase between 5- and 10-fold from normal circulating levels within 1 h after administration of LPS [42,130,131]. The initial rise in activin A slightly preceded the classical rise in TNFa, and occurred prior to the rise in ILb or IL6. As a result, activin A increased in the circulation before the three most important early pro-inflammatory cytokines, reaching a peak at 1– 2 h after LPS administration, then declining. Levels of activin B and follistatin increased in the blood several hours later, along with a secondary rise in activin A [42,131] (data not published). Generally, activin A and B levels had returned to normal or were close to normal within 24 h after LPS-treatment. Detailed investigation of this response to LPS in the mouse indicated that, unlike the increase of either TNFa or ILb, the initial rise in activin A was not due to an increase in transcriptional

Fig. 4. The putative role of the neutrophil in activin A production in acute (LPSinduced) inflammation. Neutrophils containing stored activin A leave the bone marrow in response to the inflammatory stimulus and enter the circulation and tissues, such as the lung. Inflammatory mediators, which include TNFa, stimulate the release of activin A from these cells, resulting in a rapid increase in activin A levels in the blood and target tissues. New synthesis and release of stored activin A by bone marrow stromal cells and epithelial cells in the tissues also contribute to the rapid increase in activin A, which is eventually bound by follistatin and cleared.

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block activin A activity, strongly suggests that follistatin secretion responds to the large initial increase in activin A [42]. This evidence for activin A stimulating follistatin is consistent with findings from other studies [29,134], but it is important to note that follistatin expression is known to be stimulated by several other proinflammatory cytokines, including IL1b, TNFa, and IFNg [42,135,136]. Further studies of the regulation of follistatin are required. Nevertheless, it appears that the increase in follistatin following LPS administration occurs in response to the earlier inflammatory events and serves to modulate activin activity later in the response. In the experiments where follistatin was administered prior to LPS, the early inflammatory response was also substantially altered by a reduction of TNFa and IL1b levels, as well as the absence of the secondary rise in activin A [42]. However, release of IL6 in response to LPS was augmented and temporally shifted to occur at 1 h instead of 2–3 h post-LPS treatment. Thus, modifying the bioactivity of activin released by LPS through the administration of follistatin substantially modified the entire inflammatory cascade. Most critically, pre-treatment with follistatin substantially improved the survival of mice given a lethal dose of LPS, and survival was tightly predicted by the levels of activin A produced [42]. These data indicate that blocking activin A with follistatin can reduce the severity of the acute inflammatory response. 5.3. Other inflammatory models and blocking studies Studies using knockout and transgenic mice have shown that the absence of activin or its regulatory elements modulates inflammatory and fibrotic responses in a variety of settings. These outcomes are not always intuitive: deletion of the inhibin asubunit increases systemic activin levels leading to leads to liver inflammation, cachexia and death, which can be prevented by crossing the mice with a targeted deletion of the activin type 2 receptor, ACVR2A [137], while overexpression of the bC subunit reduces activin A bioactivity and also leads to liver inflammation [12], but prevents cachexia in the inhibin a-subunit deficient mouse [13]. On the other hand, transgenic overexpression of follistatin (i.e. inhibiting activin) reduced liver inflammation, necrosis and cachexia in the mice lacking the inhibin a-subunit [138]. Such evidently disparate results serve to highlight the complexity of the various roles of activin A in cellular and immunological regulation and disease. Selectively overexpressing follistatin in the epidermis delayed wound healing but inhibited dermal scar formation in mice [139]. It is possible that activin may actually be a critical intermediate in the induction of fibrosis. Follistatin blocks the stimulatory actions of TGFb1 on a-smooth muscle actin and collagen expression in rat hepatic stellate cells, renal fibroblasts and lung fibroblasts in vitro [76,140]. This action of follistatin seems, almost certainly, to be attributable to inhibition of the activin produced by these cells in response to TGFb [77,85], because follistatin does not bind to TGFb1 or block its actions [115–117]. This leads to the intriguing hypothesis that TGFb1 stimulates fibrosis through activin acting as an essential intermediary. Treatment with exogenous follistatin has been shown to reduce inflammation and fibrosis in models of induced lung and liver damage [85,141], colitis [142], and allergic asthma [96,143]. However, interference with the anti-inflammatory or immunoregulatory actions of activin has been implicated in experimental models where follistatin administration or activin-neutralising antibodies appears to exacerbate inflammation, in a kainic acidinduced neurodegneration model [57] and in allergic asthma [97]. These apparently conflicting outcomes suggest that timing, dose and route of administration are all critical in determining the overall effects of follistatin in regulating inflammation, in

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particular. Similar caveats almost certainly will apply to the use of other activin inhibitors, such as those based on activin receptor subunits or the activin propeptide [19,144]. 6. Potential for therapeutic and diagnostics applications in disease Altogether, the existing data establish that the activins are key intermediates and regulators of inflammatory and fibrotic responses, and that blocking activin actions can ameliorate many of the deleterious effects caused by these responses. Although studies to date have been confined to animal models, it is not hard to imagine that an endogenous regulator of activin, such as follistatin, may have considerable therapeutic potential in many serious and life-threatening conditions. Modulation of activin activity by follistatin may be an intervention option in humans undergoing endotoxaemic shock, which represents one of the most common causes of death in hospital because patients respond poorly to treatment. It already has been established that higher levels of serum activin A in patients with sepsis are predictive of death [128]. It is likely that the link between activin A levels and death is related to the capacity of activin A to induce apoptosis of hepatocytes, causing liver failure [58], and B lymphocytes [106]. The concept is further supported by the studies in mice with a targeted deletion of the inhibin a subunit, which display elevated activin A levels and profound inflammation, cachexia and death, outcomes that can be prevented by blocking activin action [137,138,145,146]. Likewise, the profound disturbance of respiratory structure and function in mice over-expressing the activin A due to intra-tracheal administration of an adeno-associated viral vector containing the bA subunit sequence provides an explanation for the high mortality associated with elevated serum activin A levels found in patients in intensive care units with sepsis [144]. Indeed, numerous studies have indicated an important role for activin in serious lung diseases, such as acute lung injury/acute respiratory distress syndrome, chronic obstructive pulmonary disease, cystic fibrosis and asthma [77,81,86,97–99,144,147], inflammation and fibrosis of the liver, kidney and pancreas [76,78,141], wound healing [139], chronic inflammatory diseases like rheumatoid arthritis [73,83] or inflammatory bowel disease [142,148] and pre-eclampsia [149]. Studies in humans with these and other inflammatory and fibrotic diseases should build a convincing case for the use of follistatin as a therapeutic modality, that has already been indicated by successful studies in animals [85,96,141–143]. Further examples of pathophysiological states wherein the regulation of activin bioactivity by the use of follistatin or other activin-blocking modalities may be beneficial arise from studies of surgical trauma and resulting tissue injury. Animal models have shown that release of activin A and follistatin into the circulation are features of the response to surgical trauma [150,151]. The specific mechanisms that induce this secretion remain to be fully elucidated, but TLR signalling and the inflammatory cytokine cascade are obviously implicated [152]. Damage occurs through ischaemia–reperfusion injury, and follistatin has been shown to provide protection against ischaemia–reperfusion injury in the rat liver and kidney [153,154]. In some surgical situations, the problem may be exacerbated by the use of heparin-based anticoagulants, which have been shown to release substantial amounts of activin A and follistatin in animal and human studies [155,156]. These increments arise from the fact that follistatin and the activin–follistatin complex bind to heparan sulphate proteoglycans on the surface of cells via positively charged amino acids and the negatively charged heparin interferes with this binding [112,157]. In a recent study of cardiopulmonary bypass in sheep, it was discovered that the magnitude of the activin A and follistatin

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response to surgery could be greatly decreased by the use of a nonheparin based anti-coagulant, lepirudin [150]. Since heparin is used extensively in organ retrieval and transplant surgery, the release of activin A in such situations could be detrimental to subsequent graft function. Modulation of the activity of the activins during transplantation surgery by the use of exogenous follistatin or other inhibitors of activin and the use of non-heparin anti-coagulants may lead to improved preservation and extend the functional life of transplanted organs – this is something that should be actively explored. Finally, there is considerable potential for the use of activin, follistatin and activin/follistatin ratios as markers of disease progression, severity and management in acute and chronic inflammatory diseases, such as sepsis, pre-eclampsia, cadiovascular disease and even type 2 diabetes [128,149,158,159]. This diagnostic application depends on the establishment of clinical normal range values using sensitive, precise, highly specific assays that are not subject to interference by activin and follistatin binding interactions or other serum factors, and clearly defined and accepted standard preparations. 7. Conclusions After commencing life as regulators of reproductive processes, the activins and their binding protein, follistatin, are beginning to emerge as important diagnostic tools and therapeutic targets in a range on inflammatory, immunological and fibrotic diseases. Animal models using follistatin as an inhibitor of activin actions have demonstrated considerable promise, but there remain many questions regarding both the basic biology and potential clinical applications. One important issue will be the development of appropriate strategies to calibrate treatment to accommodate the pleiotrophic role of the activins in inflammation, immunoregulation and cell growth and development. Nonetheless, it is certain that the control of activin activity, and its consequences, represents an exciting avenue for investigation in the coming years. Acknowledgements The work of the authors described in this review has been supported by a Fellowship (to M.P.H.) and Grants from the National Health and Medical Research Council, and by the Victorian Government’s Operational Infrastructure Support Program. References [1] Ling N, Ying SY, Ueno N, Shimasaki S, Esch F, Hotta M, et al. Pituitary FSH is released by a heterodimer of the b-subunits from the two forms of inhibin. Nature 1986;321:779–82. [2] Yu J, Shao LE, Lemas V, Yu AL, Vaughan J, Rivier J, et al. Importance of FSHreleasing protein and inhibin in erythrodifferentiation. Nature 1987;330: 765–7. [3] Broxmeyer HE, Lu L, Cooper S, Schwall RH, Mason AJ, Nikolics K. Selective and indirect modulation of human multipotential and erythroid hematopoietic progenitor cell proliferation by recombinant human activin and inhibin. Proceedings of the National Academy of Sciences of the United States of America 1988;85:9052–6. [4] Hedger MP, Drummond AE, Robertson DM, Risbridger GP, de Kretser DM. Inhibin and activin regulate [3H]thymidine uptake by rat thymocytes and 3T3 cells in vitro. Molecular and Cellular Endocrinology 1989;61:133–8. [5] Hedger MP, Winnall WR, Phillips DJ, de Kretser DM. The regulation and functions of activin and follistatin in inflammation and immunity. Vitamins and Hormones 2011;85:255–97. [6] Brosh N, Sternberg D, Honigwachs-Sha’anani J, Lee BC, Shav-Tal Y, Tzehoval E. The plasmacytoma growth inhibitor restrictin-P is an antagonist of interleukin 6 and interleukin 11. Identification as a stroma-derived activin A. Journal of Biological Chemistry 1995;270:29594–600. [7] Eto Y, Tsuji T, Takezawa M, Takano S, Yokogawa Y, Shibai H. Purification and characterization of erythroid differentiation factor (EDF) isolated from human leukemia cell line THP-1. Biochemical and Biophysical Research Communications 1987;142:1095–103.

[8] Mason AJ, Niall HD, Seeburg PH. Structure of two human ovarian inhibins. Biochemical and Biophysical Research Communications 1986;135: 957–64. [9] Thompson TB, Cook RW, Chapman SC, Jardetzky TS, Woodruff TK. Beta A versus beta B: is it merely a matter of expression? Molecular and Cellular Endocrinology 2004;225:9–17. [10] Fang J, Wang SQ, Smiley E, Bonadio J. Genes coding for mouse activin bC and bE are closely linked and exhibit a liver-specific expression pattern in adult tissues. Biochemical and Biophysical Research Communications 1997;231: 655–61. [11] O’Bryan MK, Sebire KL, Gerdprasert O, Hedger MP, Hearn MT, de Kretser DM. Cloning and regulation of the rat activin bE subunit. Journal of Molecular Endocrinology 2000;24:409–18. [12] Gold E, Jetly N, O’Bryan MK, Meachem S, Srinivasan D, Behuria S, et al. Activin C antagonizes activin A in vitro and overexpression leads to pathologies in vivo. American Journal of Pathology 2009;174:184–95. [13] Gold E, Marino FE, Harrison C, Makanji Y, Risbridger G. Activin-bc reduces reproductive tumour progression and abolishes cancer-associated cachexia in inhibin deficient mice. Journal of Pathology; in press. [14] Hashimoto O, Ushiro Y, Sekiyama K, Yamaguchi O, Yoshioka K, Mutoh K, et al. Impaired growth of pancreatic exocrine cells in transgenic mice expressing human activin bE subunit. Biochemical and Biophysical Research Communications 2006;341:416–24. [15] Forage RG, Ring JM, Brown RW, McInerney BV, Cobon GS, Gregson RP, et al. Cloning and sequence analysis of cDNA species coding for the two subunits of inhibin from bovine follicular fluid. Proceedings of the National Academy of Sciences of the United States of America 1986;83:3091–5. [16] Daopin S, Piez KA, Ogawa Y, Davies DR. Crystal structure of transforming growth factor-b2: an unusual fold for the superfamily. Science 1992;257: 369–73. [17] Gray AM, Mason AJ. Requirement for activin A and transforming growth factor-b1 pro-regions in homodimer assembly. Science 1990;247:1328–30. [18] Huylebroeck D, Van Nimmen K, Waheed A, von Figura K, Marmenout A, Fransen L, et al. Expression and processing of the activin-A/erythroid differentiation factor precursor: a member of the transforming growth factor-b superfamily. Molecular Endocrinology 1990;4:1153–65. [19] Makanji Y, Walton KL, Chan KL, Gregorevic P, Robertson DM, Harrison CA. Generation of a specific activin antagonist by modification of the activin A propeptide. Endocrinology 2011;152:3758–68. [20] Walton KL, Makanji Y, Robertson DM, Harrison CA. The synthesis and secretion of inhibins. Vitamins and Hormones 2011;85:149–84. [21] Ludlow H, Phillips DJ, Myers M, McLachlan RI, de Kretser DM, Allan CA, et al. A new ‘total’ activin B enzyme-linked immunosorbent assay (ELISA): development and validation for human samples. Clinical Endocrinology 2009;71: 867–73. [22] Knight PG, Muttukrishna S, Groome NP. Development and application of a two-site enzyme immunoassay for the determination of ‘total’ activin-A concentrations in serum and follicular fluid. Journal of Endocrinology 1996;148:267–79. [23] Meunier H, Rivier C, Evans RM, Vale W. Gonadal and extragonadal expression of inhibin a, bA, and bB subunits in various tissues predicts diverse functions. Proceedings of the National Academy of Sciences of the United States of America 1988;85:247–51. [24] Schneider O, Nau R, Michel U. Comparative analysis of follistatin-, activin beta A- and activin beta B-mRNA steady-state levels in diverse porcine tissues by multiplex S1 nuclease analysis. European Journal of Endocrinology/European Federation of Endocrine Societies 2000;142:537–44. [25] Wu H, Chen Y, Winnall WR, Phillips DJ, Hedger MP. Acute regulation of activin A and its binding protein, follistatin, in serum and tissues following lipopolysaccharide treatment of adult male mice. American Journal of Physiology Regulatory Integrative and Comparative Physiology 2012;303: R665–75. [26] Winnall WR, Wu H, Sarraj MA, Rogers PA, de Kretser DM, Girling JE, et al. Expression patterns of activin, inhibin and follistatin variants in the adult male mouse reproductive tract suggest important roles in the epididymis and vas deferens. Reproduction, Fertility & Development; in press. [27] Era¨maa M, Hurme M, Stenman UH, Ritvos O. Activin A/erythroid differentiation factor is induced during human monocyte activation. Journal of Experimental Medicine 1992;176:1449–52. [28] Okuma Y, O’Connor AE, Muir JA, Stanton PG, de Kretser DM, Hedger MP. Regulation of activin A and inhibin B secretion by inflammatory mediators in adult rat Sertoli cell cultures. Journal of Endocrinology 2005;187:125–34. [29] Wilson KM, Smith AI, Phillips DJ. Stimulatory effects of lipopolysaccharide on endothelial cell activin and follistatin. Molecular and Cellular Endocrinology 2006;253:30–5. [30] Robson NC, Phillips DJ, McAlpine T, Shin A, Svobodova S, Toy T, et al. ActivinA: a novel dendritic cell-derived cytokine that potently attenuates CD40 ligand-specific cytokine and chemokine production. Blood 2008;111: 2733–43. [31] Yamashita T, Takahashi S, Ogata E. Expression of activin A/erythroid differentiation factor in murine bone marrow stromal cells. Blood 1992;79:304–7. [32] Funaba M, Ikeda T, Ogawa K, Abe M. Calcium-regulated expression of activin A in RBL-2H3 mast cells. Cellular Signalling 2003;15:605–13. [33] Ardekani AM, Romanelli JC, Mayo KE. Structure of the rat inhibin and activin bA-subunit gene and regulation in an ovarian granulosa cell line. Endocrinology 1998;139:3271–9.

M.P. Hedger, D.M. de Kretser / Cytokine & Growth Factor Reviews 24 (2013) 285–295 [34] Tanimoto K, Yoshida E, Mita S, Nibu Y, Murakami K, Fukamizu A. Human activin bA gene, Identification of novel 50 exon, functional promoter, and enhancers. Journal of Biological Chemistry 1996;271:32760–69. [35] Dolter KE, Palyash JC, Shao LE, Yu J. Analysis of activin A gene expression in human bone marrow stromal cells. Journal of Cellular Biochemistry 1998;70:8–21. [36] Yoshida E, Tanimoto K, Murakami K, Fukamizu A. Isolation and characterization of 50 -regulatory region of mouse activin bA subunit gene. Biochemistry and Molecular Biology International 1998;44:325–32. [37] Ogawa K, Funaba M, Chen Y, Tsujimoto M, Activin A. functions as a Th2 cytokine in the promotion of the alternative activation of macrophages. Journal of Immunology 2006;177:6787–94. [38] Thompson DA, Cronin CN, Martin F. Genomic cloning and sequence analyses of the bovine a-bA- and bB-inhibin/activin genes. Identification of transcription factor AP-2-binding sites in the 50 -flanking regions by DNase I footprinting. European Journal of Biochemistry 1994;226:751–64. [39] Takahashi S, Uchimaru K, Harigaya K, Asano S, Yamashita T. Tumor necrosis factor and interleukin-1 induce activin A gene expression in a human bone marrow stromal cell line. Biochemical and Biophysical Research Communications 1992;188:310–7. [40] Shao L, Frigon Jr NL, Sehy DW, Yu AL, Lofgren J, Schwall R, et al. Regulation of production of activin A in human marrow stromal cells and monocytes. Experimental Hematology 1992;20:1235–42. [41] Okuma Y, Saito K, O’Connor AE, Phillips DJ, de Kretser DM, Hedger MP. Reciprocal regulation of activin A and inhibin B by interleukin-1 (IL-1) and follicle-stimulating hormone (FSH) in rat Sertoli cells in vitro. Journal of Endocrinology 2005;185:99–110. [42] Jones KL, Mansell A, Patella S, Scott BJ, Hedger MP, de Kretser DM, et al. Activin A is a critical component of the inflammatory response, and its binding protein, follistatin, reduces mortality in endotoxemia. Proceedings of the National Academy of Sciences of the United States of America 2007;104:16239–44. [43] Walton KL, Makanji Y, Wilce MC, Chan KL, Robertson DM, Harrison CA. A common biosynthetic pathway governs the dimerization and secretion of inhibin and related transforming growth factor b (TGFb) ligands. Journal of Biological Chemistry 2009;284:9311–20. [44] Dykema JC, Mayo KE. Two messenger ribonucleic acids encoding the common bB-chain of inhibin and activin have distinct 50 -initiation sites and are differentially regulated in rat granulosa cells. Endocrinology 1994;135: 702–11. [45] Feng ZM, Bardin CW, Chen CL. Characterization and regulation of testicular inhibin b-subunit mRNA. Molecular Endocrinology 1989;3:939–48. [46] Tsuchida K, Nakatani M, Uezumi A, Murakami T, Cui X. Signal transduction pathway through activin receptors as a therapeutic target of musculoskeletal diseases and cancer. Endocrine Journal 2008;55:11–21. [47] de Caestecker M. The transforming growth factor-b superfamily of receptors. Cytokine and Growth Factor Reviews 2004;15:1–11. [48] Bernard DJ, Lee KB, Santos MM, Activin B. can signal through both ALK4 and ALK7 in gonadotrope cells. Reproductive Biology and Endocrinology 2006;4: 52. [49] Tsuchida K, Nakatani M, Yamakawa N, Hashimoto O, Hasegawa Y, Sugino H. Activin isoforms signal through type I receptor serine/threonine kinase ALK7. Molecular and Cellular Endocrinology 2004;220:59–65. [50] Mathews LS, Vale WW. Expression cloning of an activin receptor, a predicted transmembrane serine kinase. Cell 1991;65:973–82. [51] ten Dijke P, Hill CS. New insights into TGF-b-Smad signalling. Trends in Biochemical Sciences 2004;29:265–73. [52] Heldin CH, Landstro¨m M, Moustakas A. Mechanism of TGF-b signaling to growth arrest, apoptosis, and epithelial-mesenchymal transition. Current Opinion in Cell Biology 2009;21:166–76. [53] Huang HM, Chiou HY, Chang JL, Activin A. induces erythroid gene expressions and inhibits mitogenic cytokine-mediated K562 colony formation by activating p38 MAPK. Journal of Cellular Biochemistry 2006;98:789–97. [54] Zhang L, Deng M, Parthasarathy R, Wang L, Mongan M, Molkentin JD, et al. MEKK1 transduces activin signals in keratinocytes to induce actin stress fiber formation and migration. Molecular and Cellular Biology 2005;25:60–5. [55] Kaneda H, Arao T, Matsumoto K, De Velasco MA, Tamura D, Aomatsu K, et al. Activin A inhibits vascular endothelial cell growth and suppresses tumour angiogenesis in gastric cancer. British Journal of Cancer 2011;105:1210–7. [56] Thomsen G, Woolf T, Whitman M, Sokol S, Vaughan J, Vale W, et al. Activins are expressed early in Xenopus embryogenesis and can induce axial mesoderm and anterior structures. Cell 1990;63:485–93. [57] Abdipranoto-Cowley A, Park JS, Croucher D, Daniel J, Henshall S, Galbraith S, et al. Activin A is essential for neurogenesis following neurodegeneration. Stem Cells 2009;27:1330–46. [58] Schwall RH, Robbins K, Jardieu P, Chang L, Lai C, Terrell TG. Activin induces cell death in hepatocytes in vivo and in vitro. Hepatology 1993;18:347–56. [59] Yamashita N, Nakajima T, Takahashi H, Kaneoka H, Mizushima Y, Sakane T. Effects of activin A on IgE synthesis and cytokine production by human peripheral mononuclear cells. Clinical and Experimental Immunology 1993;94:214–9. [60] Nu¨sing RM, Barsig J. Induction of prostanoid, nitric oxide, and cytokine formation in rat bone marrow derived macrophages by activin A. British Journal of Pharmacology 1999;127:919–26. [61] Murase Y, Okahashi N, Koseki T, Itoh K, Udagawa N, Hashimoto O, et al. Possible involvement of protein kinases and Smad2 signaling pathways on

[62]

[63]

[64] [65]

[66]

[67]

[68]

[69]

[70]

[71]

[72]

[73]

[74]

[75]

[76]

[77]

[78]

[79]

[80]

[81]

[82]

[83]

[84]

[85]

[86]

[87]

293

osteoclast differentiation enhanced by activin A. Journal of Cellular Physiology 2001;188:236–42. Sugatani T, Alvarez UM, Hruska KA. Activin A stimulates IkB-a/NFkB and RANK expression for osteoclast differentiation, but not AKT survival pathway in osteoclast precursors. Journal of Cellular Biochemistry 2003;90:59–67. Ohguchi M, Yamato K, Ishihara Y, Koide M, Ueda N, Okahashi N, et al. Activin A regulates the production of mature interleukin-1b and interleukin-1 receptor antagonist in human monocytic cells. Journal of Interferon and Cytokine Research 1998;18:491–8. Cuschieri J, Bulger E, Grinsell R, Garcia I, Maier RV. Insulin regulates macrophage activation through activin A. Shock 2008;29:285–90. Sugama S, Takenouchi T, Kitani H, Fujita M, Hashimoto M. Activin as an antiinflammatory cytokine produced by microglia. Journal of Neuroimmunology 2007;192:31–9. Wang SY, Tai GX, Zhang PY, Mu DP, Zhang XJ, Liu ZH. Inhibitory effect of activin A on activation of lipopolysaccharide-stimulated mouse macrophage RAW264.7 cells. Cytokine 2008;42:85–91. Wang Y, Cui X, Tai G, Ge J, Li N, Chen F, et al. A critical role of activin A in maturation of mouse peritoneal macrophages in vitro and in vivo. Cellular & Molecular Immunology 2009;6:387–92. Wilms H, Schwark T, Brandenburg LO, Sievers J, Dengler R, Deuschl G, et al. Regulation of activin A synthesis in microglial cells: pathophysiological implications for bacterial meningitis. Journal of Neuroscience Research 2010;88:16–23. Zhang XJ, Li Y, Tai GX, Xu GY, Zhang PY, Yang Y, et al. Effects of activin A on the activities of the mouse peritoneal macrophages. Cellular & Molecular Immunology 2005;2:63–7. Zhou J, Tai G, Liu H, Ge J, Feng Y, Chen F, et al. Activin A. down-regulates the phagocytosis of lipopolysaccharide-activated mouse peritoneal macrophages in vitro and in vivo. Cellular Immunology 2009;255:69–75. Hedger MP, Phillips DJ, de Kretser DM. Divergent cell-specific effects of activin-A on thymocyte proliferation stimulated by phytohemagglutinin, and interleukin 1b or interleukin 6 in vitro. Cytokine 2000;12:595–602. Russell CE, Hedger MP, Brauman JN, de Kretser DM, Phillips DJ. Activin A regulates growth and acute phase proteins in the human liver cell line, HepG2. Molecular and Cellular Endocrinology 1999;148:129–36. Yu EW, Dolter KE, Shao LE, Yu J. Suppression of IL-6 biological activities by activin A and implications for inflammatory arthropathies. Clinical and Experimental Immunology 1998;112:126–32. Ohga E, Matsuse T, Teramoto S, Katayama H, Nagase T, Fukuchi Y, et al. Effects of activin A on proliferation and differentiation of human lung fibroblasts. Biochemical and Biophysical Research Communications 1996;228:391–6. Ota F, Maeshima A, Yamashita S, Ikeuchi H, Kaneko Y, Kuroiwa T, et al. Activin A induces cell proliferation of fibroblast-like synoviocytes in rheumatoid arthritis. Arthritis and Rheumatism 2003;48:2442–9. Yamashita S, Maeshima A, Kojima I, Nojima Y. Activin A is a potent activator of renal interstitial fibroblasts. Journal of the American Society of Nephrology 2004;15:91–101. Karagiannidis C, Hense G, Martin C, Epstein M, Ru¨ckert B, Mantel PY, et al. Activin A is an acute allergen-responsive cytokine and provides a link to TGFb-mediated airway remodeling in asthma. Journal of Allergy and Clinical Immunology 2006;117:111–8. Ohnishi N, Miyata T, Ohnishi H, Yasuda H, Tamada K, Ueda N, et al. Activin A is an autocrine activator of rat pancreatic stellate cells: potential therapeutic role of follistatin for pancreatic fibrosis. Gut 2003;52:1487–93. Karger A, Fitzner B, Brock P, Sparmann G, Emmrich J, Liebe S, et al. Molecular insights into connective tissue growth factor action in rat pancreatic stellate cells. Cellular Signalling 2008;20:1865–72. Murakami M, Ikeda T, Saito T, Ogawa K, Nishino Y, Nakaya K, et al. Transcriptional regulation of plasminogen activator inhibitor-1 by transforming growth factor-b, activin A and microphthalmia-associated transcription factor. Cellular Signalling 2006;18:256–65. Yndestad A, Larsen KO, Øie E, Ueland T, Smith C, Halvorsen B, et al. Elevated levels of activin A in clinical and experimental pulmonary hypertension. Journal of Applied Physiology 2009;106:1356–64. Gaedeke J, Boehler T, Budde K, Neumayer HH, Peters H. Glomerular activin A overexpression is linked to fibrosis in anti-Thy1 glomerulonephritis. Nephrology Dialysis Transplantation 2005;20:319–28. Gribi R, Tanaka T, Harper-Summers R, Yu J. Expression of activin A in inflammatory arthropathies. Molecular and Cellular Endocrinology 2001;180: 163–7. Era¨maa M, Ritvos O. Transforming growth factor-beta 1 and -beta 2 induce inhibin and activin beta B-subunit messenger ribonucleic acid levels in cultured human granulosa-luteal cells. Fertility and Sterility 1996;65: 954–60. Aoki F, Kurabayashi M, Hasegawa Y, Kojima I. Attenuation of bleomycininduced pulmonary fibrosis by follistatin. American Journal of Respiratory and Critical Care Medicine 2005;172:713–20. Hardy CL, Lemasurier JS, Olsson F, Dang T, Yao J, Yang M, et al. IL-13 regulates secretion of the tumor growth factor-b superfamily cytokine activin A in allergic airway inflammation. American Journal of Respiratory Cell and Molecular Biology 2010;42:667–75. Pawlowski JE, Taylor DS, Valentine M, Hail ME, Ferrer P, Kowala MC, et al. Stimulation of activin A expression in rat aortic smooth muscle cells by thrombin and angiotensin II correlates with neointimal formation in vivo. Journal of Clinical Investigation 1997;100:639–48.

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[88] Reis FM, Luisi S, Florio P, Degrassi A, Petraglia F. Corticotropin-releasing factor, urocortin and endothelin-1 stimulate activin A release from cultured human placental cells. Placenta 2002;23:522–5. [89] Musso T, Scutera S, Vermi W, Daniele R, Fornaro M, Castagnoli C, et al. Activin A induces Langerhans cell differentiation in vitro and in human skin explants. PLoS ONE 2008;3:e3271. [90] Salogni L, Musso T, Bosisio D, Mirolo M, Jala VR, Haribabu B, et al. Activin A induces dendritic cell migration through the polarized release of CXC chemokine ligands 12 and 14. Blood 2009;113:5848–56. [91] Scutera S, Riboldi E, Daniele R, Elia AR, Fraone T, Castagnoli C, et al. Production and function of activin A in human dendritic cells. European Cytokine Network 2008;19:60–8. [92] Segerer SE, Mu¨ller N, Brandt J, Kapp M, Dietl J, Reichardt HM, et al. The glycoprotein-hormones activin A and inhibin A interfere with dendritic cell maturation. Reproductive Biology and Endocrinology 2008;6:17. [93] Robson NC, Wei H, McAlpine T, Kirkpatrick N, Cebon J, Maraskovsky E. Activin-A attenuates several human natural killer cell functions. Blood 2009;113:3218–25. [94] Huber S, Stahl FR, Schrader J, Lu¨th S, Presser K, Carambia A, et al. Activin a promotes the TGF-b-induced conversion of CD4+CD25 T cells into Foxp3+ induced regulatory T cells. Journal of Immunology 2009;182:4633–40. [95] Abe M, Shintani Y, Eto Y, Harada K, Kosaka M, Matsumoto T. Potent induction of activin A secretion from monocytes and bone marrow stromal fibroblasts by cognate interaction with activated T cells. Journal of Leukocyte Biology 2002;72:347–52. [96] Hardy CL, O’Connor AE, Yao J, Sebire K, de Kretser DM, Rolland JM, et al. Follistatin is a candidate endogenous negative regulator of activin A in experimental allergic asthma. Clinical and Experimental Allergy 2006;36: 941–50. [97] Semitekolou M, Alissafi T, Aggelakopoulou M, Kourepini E, Kariyawasam HH, Kay AB, et al. Activin-A induces regulatory T cells that suppress T helper cell immune responses and protect from allergic airway disease. Journal of Experimental Medicine 2009;206:1769–85. [98] Jones CP, Gregory LG, Causton B, Campbell GA, Lloyd CM. Activin A and TGF-b promote TH9 cell-mediated pulmonary allergic pathology. Journal of Allergy and Clinical Immunology. doi:S0091-6749(11)02934-4 [pii], in press. [99] Kariyawasam HH, Pegorier S, Barkans J, Xanthou G, Aizen M, Ying S, et al. Activin and transforming growth factor-b signaling pathways are activated after allergen challenge in mild asthma. Journal of Allergy and Clinical Immunology 2009;124:454–62. [100] Wohlfahrt JG, Kunzmann S, Menz G, Kneist W, Akdis CA, Blaser K, et al. T cell phenotype in allergic asthma and atopic dermatitis. International Archives of Allergy and Immunology 2003;131:272–82. [101] Ogawa K, Funaba M, Tsujimoto M. A dual role of activin A in regulating immunoglobulin production of B cells. Journal of Leukocyte Biology 2008;83:1451–8. [102] Famulski KS, Sis B, Billesberger L, Halloran PF. Interferon-g and donor MHC class I control alternative macrophage activation and activin expression in rejecting kidney allografts: a shift in the Th1–Th2 paradigm. American Journal of Transplantation 2008;8:547–56. [103] Sierra-Filardi E, Puig-Kro¨ger A, Blanco FJ, Nieto C, Bragado R, Palomero MI, et al. Activin A skews macrophage polarization by promoting a proinflammatory phenotype and inhibiting the acquisition of anti-inflammatory macrophage markers. Blood 2011;117:5092–101. [104] Petraglia F, Sacerdote P, Cossarizza A, Angioni S, Genazzani AD, Franceschi C, et al. Inhibin and activin modulate human monocyte chemotaxis and human lymphocyte interferon-g production. Journal of Clinical Endocrinology and Metabolism 1991;72:496–502. [105] Nishihara T, Okahashi N, Ueda N. Activin A induces apoptotic cell death. Biochemical and Biophysical Research Communications 1993;197: 985–91. [106] Zipori D, Barda-Saad M. Role of activin A in negative regulation of normal and tumor B lymphocytes. Journal of Leukocyte Biology 2001;69:867–73. [107] Shimasaki S, Koga M, Esch F, Mercado M, Cooksey K, Koba A, et al. Porcine follistatin gene structure supports two forms of mature follistatin produced by alternative splicing. Biochemical and Biophysical Research Communications 1988;152:717–23. [108] Nakamura T, Takio K, Eto Y, Shibai H, Titani K, Sugino H. Activin-binding protein from rat ovary is follistatin. Science 1990;247:836–8. [109] Sugino K, Kurosawa N, Nakamura T, Takio K, Shimasaki S, Ling N, et al. Molecular heterogeneity of follistatin, an activin-binding protein. Higher affinity of the carboxyl-terminal truncated forms for heparan sulfate proteoglycans on the ovarian granulosa cell. Journal of Biological Chemistry 1993;268:15579–87. [110] Schneyer A, Schoen A, Quigg A, Sidis Y. Differential binding and neutralization of activins A and B by follistatin and follistatin like-3 (FSTL-3/FSRP/FLRG). Endocrinology 2003;144:1671–4. [111] Inouye S, Guo Y, Ling N, Shimasaki S. Site-specific mutagenesis of human follistatin. Biochemical and Biophysical Research Communications 1991;179: 352–8. [112] Lerch TF, Shimasaki S, Woodruff TK, Jardetzky TS. Structural and biophysical coupling of heparin and activin binding to follistatin isoform functions. Journal of Biological Chemistry 2007;282:15930–39. [113] Hashimoto O, Nakamura T, Shoji H, Shimasaki S, Hayashi Y, Sugino H. A novel role of follistatin, an activin-binding protein, in the inhibition of activin action in rat pituitary cells. Endocytotic degradation of activin and its

[114]

[115]

[116]

[117]

[118]

[119]

[120]

[121]

[122] [123]

[124]

[125]

[126]

[127]

[128]

[129]

[130]

[131]

[132]

[133]

[134]

[135]

[136]

[137]

[138]

[139]

acceleration by follistatin associated with cell-surface heparan sulfate. Journal of Biological Chemistry 1997;272:13835–42. Amthor H, Nicholas G, McKinnell I, Kemp CF, Sharma M, Kambadur R, et al. Follistatin complexes Myostatin and antagonises. Myostatin-mediated inhibition of myogenesis. Developmental Biology 2004;270:19–30. Harrington AE, Morris-Triggs SA, Ruotolo BT, Robinson CV, Ohnuma S, Hyvo¨nen M. Structural basis for the inhibition of activin signalling by follistatin. EMBO Journal 2006;25:1035–45. Iemura S, Yamamoto TS, Takagi C, Uchiyama H, Natsume T, Shimasaki S, et al. Direct binding of follistatin to a complex of bone-morphogenetic protein and its receptor inhibits ventral and epidermal cell fates in early Xenopus embryo. Proceedings of the National Academy of Sciences of the United States of America 1998;95:9337–42. Nogai H, Rosowski M, Grun J, Rietz A, Debus N, Schmidt G, et al. Follistatin antagonizes transforming growth factor-b3-induced epithelial-mesenchymal transition in vitro: implications for murine palatal development supported by microarray analysis. Differentiation 2007;76:404–16. Sidis Y, Tortoriello DV, Holmes WE, Pan Y, Keutmann HT, Schneyer AL. Follistatin-related protein and follistatin differentially neutralize endogenous vs. exogenous activin. Endocrinology 2002;143:1613–24. Niemuller CA, Randall KJ, Webb DJ, Gonias SL, LaMarre J. a2-macroglobulin conformation determines binding affinity for activin A and plasma clearance of activin A/a2-macroglobulin complex. Endocrinology 1995;136:5343–9. Vale W, Wiater E, Gray P, Harrison C, Bilezikjian L, Choe S. Activins and inhibins and their signaling. Annals of the New York Academy of Sciences 2004;1038:142–7. Bilezikjian LM, Vaughan JM, Vale WW. Characterization and the regulation of inhibin/activin subunit proteins of cultured rat anterior pituitary cells. Endocrinology 1993;133:2545–53. Pangas SA, Woodruff TK. Production and purification of recombinant human inhibin and activin. Journal of Endocrinology 2002;172:199–210. Muenster U, Harrison CA, Donaldson C, Vale W, Fischer WH. An activin-A/C chimera exhibits activin and myostatin antagonistic properties. Journal of Biological Chemistry 2005;280:36626–32. Mellor SL, Ball EM, O’Connor AE, Ethier JF, Cranfield M, Schmitt JF, et al. Activin bC-subunit heterodimers provide a new mechanism of regulating activin levels in the prostate. Endocrinology 2003;144:4410–9. Barbara NP, Wrana JL, Letarte M. Endoglin is an accessory protein that interacts with the signaling receptor complex of multiple members of the transforming growth factor-b superfamily. Journal of Biological Chemistry 1999;274:584–94. Gray PC, Harrison CA, Vale W. Cripto forms a complex with activin and type II activin receptors and can block activin signaling. Proceedings of the National Academy of Sciences of the United States of America 2003;100:5193–8. Onichtchouk D, Chen YG, Dosch R, Gawantka V, Delius H, Massague J, et al. Silencing of TGF-beta signalling by the pseudoreceptor BAMBI. Nature 1999;401:480–5. Michel U, Ebert S, Phillips D, Nau R. Serum concentrations of activin and follistatin are elevated and run in parallel in patients with septicemia. European Journal of Endocrinology/European Federation of Endocrine Societies 2003;148:559–64. Phillips DJ, de Kretser DM, Hedger MP. Activin and related proteins in inflammation: not just interested bystanders. Cytokine and Growth Factor Reviews 2009;20:153–64. Jones KL, Brauman JN, Groome NP, de Kretser DM, Phillips DJ. Activin A release into the circulation is an early event in systemic inflammation and precedes the release of follistatin. Endocrinology 2000;141:1905–8. Jones KL, de Kretser DM, Clarke IJ, Scheerlinck JP, Phillips DJ. Characterisation of the rapid release of activin A following acute lipopolysaccharide challenge in the ewe. Journal of Endocrinology 2004;182:69–80. Chen Y, Wu H, Winnall WR, Loveland KL, Makanji Y, Phillips DJ, et al. Tumour necrosis factor-a stimulates human neutrophils to release preformed activin A. Immunology and Cell Biology 2011;89:889–96. Wu H, Chen Y, Winnall WR, Phillips DJ, Hedger MP. Regulation of activin A release from murine bone marrow-derived neutrophil precursors by tumour necrosis factor-a and insulin. Cytokine 2013;61:199–204. Blount AL, Vaughan JM, Vale WW, Bilezikjian LM. A Smad-binding element in intron 1 participates in activin-dependent regulation of the follistatin gene. Journal of Biological Chemistry 2008;283:7016–26. Abe M, Shintani Y, Eto Y, Harada K, Fujinaka Y, Kosaka M, et al. Interleukin-1 b enhances and interferon-gamma suppresses activin A actions by reciprocally regulating activin A and follistatin secretion from bone marrow stromal fibroblasts. Clinical and Experimental Immunology 2001;126:64–8. Keelan JA, Zhou RL, Evans LW, Groome NP, Mitchell MD. Regulation of activin A, inhibin A, and follistatin production in human amnion and choriodecidual explants by inflammatory mediators. Journal of the Society for Gynecologic Investigation 2000;7:291–6. Coerver KA, Woodruff TK, Finegold MJ, Mather J, Bradley A, Matzuk MM. Activin signaling through activin receptor type II causes the cachexia-like symptoms in inhibin-deficient mice. Molecular Endocrinology 1996;10: 534–43. Cipriano SC, Chen L, Kumar TR, Matzuk MM. Follistatin is a modulator of gonadal tumor progression and the activin-induced wasting syndrome in inhibin-deficient mice. Endocrinology 2000;141:2319–27. Bamberger C, Scha¨rer A, Antsiferova M, Tychsen B, Pankow S, Mu¨ller M, et al. Activin controls skin morphogenesis and wound repair predominantly via

M.P. Hedger, D.M. de Kretser / Cytokine & Growth Factor Reviews 24 (2013) 285–295

[140]

[141]

[142]

[143]

[144]

[145]

[146]

[147]

[148]

[149]

[150]

[151]

[152]

[153]

stromal cells and in a concentration-dependent manner via keratinocytes. American Journal of Pathology 2005;167:733–47. Wada W, Kuwano H, Hasegawa Y, Kojima I. The dependence of transforming growth factor-b-induced collagen production on autocrine factor activin A in hepatic stellate cells. Endocrinology 2004;145:2753–9. Patella S, Phillips DJ, Tchongue J, de Kretser DM, Sievert W. Follistatin attenuates early liver fibrosis: effects on hepatic stellate cell activation and hepatocyte apoptosis. American Journal of Physiology Gastrointestinal and Liver Physiology 2006;290:G137–44. Dohi T, Ejima C, Kato R, Kawamura YI, Kawashima R, Mizutani N, et al. Therapeutic potential of follistatin for colonic inflammation in mice. Gastroenterology 2005;128:411–23. Hardy CL, Nguyen HA, Mohamud R, Yao J, Oh DY, Plebanski M, et al. The activin A antagonist follistatin inhibits asthmatic airway remodelling. Thorax 2013;68:9–18. Apostolou E, Stavropoulos A, Sountoulidis A, Xirakia C, Giaglis S, Protopapadakis E, et al. Activin-A over-expression in the murine lung causes pathology that simulates Acute Respiratory Distress Syndrome (ARDS). American Journal of Respiratory and Critical Care Medicine; doi:201105-0784OC [pii]; in press. Matzuk MM, Finegold MJ, Mather JP, Krummen L, Lu H, Bradley A. Development of cancer cachexia-like syndrome and adrenal tumors in inhibindeficient mice. Proceedings of the National Academy of Sciences of the United States of America 1994;91:8817–21. Zhou X, Wang JL, Lu J, Song Y, Kwak KS, Jiao Q, et al. Reversal of cancer cachexia and muscle wasting by ActRIIB antagonism leads to prolonged survival. Cell 2010;142:531–43. Van Pottelberge GR, Bracke KR, Demedts IK, De Rijck K, Reinartz SM, van Drunen CM, et al. Selective accumulation of langerhans-type dendritic cells in small airways of patients with COPD. Respiratory Research 2010;11:35. Hu¨bner G, Brauchle M, Gregor M, Werner S. Activin A: a novel player and inflammatory marker in inflammatory bowel disease? Laboratory Investigation 1997;77:311–8. Spencer K, Yu CK, Savvidou M, Papageorghiou AT, Nicolaides KH. Prediction of pre-eclampsia by uterine artery Doppler ultrasonography and maternal serum pregnancy-associated plasma protein-A, free beta-human chorionic gonadotropin, activin A and inhibin A at 22 + 0 to 24 + 6 weeks’ gestation. Ultrasound in Obstetrics and Gynecology 2006;27:658–63. Chen Y, Phillips DJ, McMillan J, Bedford P, Goldstein J, Wu H, et al. Pattern of activin A and follistatin release in a sheep model of cardiopulmonary bypass. Cytokine 2011;54:154–60. Phillips DJ, Hedger MP, McFarlane JR, Klein R, Clarke IJ, Tilbrook AJ, et al. Follistatin concentrations in male sheep increase following sham castration/ castration or injection of interleukin-1b. Journal of Endocrinology 1996;151: 119–24. Hoth JJ, Wells JD, Brownlee NA, Hiltbold EM, Meredith JW, McCall CE, et al. Toll-like receptor 4-dependent responses to lung injury in a murine model of pulmonary contusion. Shock 2009;31:376–81. Kanamoto M, Shimada M, Morine Y, Yoshizumi T, Imura S, Ikegami T, et al. Beneficial effects of follistatin in hepatic ischemia-reperfusion injuries in rats. Digestive Diseases and Sciences 2011;56:1075–81.

295

[154] Maeshima A, Zhang YQ, Nojima Y, Naruse T, Kojima I. Involvement of the activin–follistatin system in tubular regeneration after renal ischemia in rats. Journal of the American Society of Nephrology 2001;12:1685–95. [155] Jones KL, De Kretser DM, Phillips DJ. Effect of heparin administration to sheep on the release profiles of circulating activin A and follistatin. Journal of Endocrinology 2004;181:307–14. [156] Phillips DJ, Jones KL, McGaw DJ, Groome NP, Smolich JJ, Parsson H, et al. Release of activin and follistatin during cardiovascular procedures is largely due to heparin administration. Journal of Clinical Endocrinology and Metabolism 2000;85:2411–5. [157] Nakamura T, Sugino K, Titani K, Sugino H. Follistatin, an activin-binding protein, associates with heparan sulfate chains of proteoglycans on follicular granulosa cells. Journal of Biological Chemistry 1991;266:19432–37. [158] Ueland T, Aukrust P, Aakhus S, Smith C, Endresen K, Birkeland KI, et al. Activin A and cardiovascular disease in type 2 diabetes mellitus. Diabetes & Vascular Disease Research 2012;9:234–7. [159] Wu H, Wu M, Chen Y, Allan CA, Phillips DJ, Hedger MP. Correlation between blood activin levels and clinical parameters of type 2 diabetes. Experimental Diabetes Research; in press.

M.P. Hedger is a Senior Research Fellow of the National Health and Medical Research Council and a Senior Scientist at the Monash Institute of Medical Research, Monash University in Melbourne, Australia. He received his PhD from Monash University in 1984 for studies in the field of male reproduction, and has published extensively on the regulation of steroidogenesis, cytokine action and immunity in the testis. His current interests lie at the interface between the immune system and the male reproductive tract and the biology of activin and follistatin in reproduction and inflammatory disease.

D.M. de Kretser is a Monash Distinguished Professor at Monash University, Melbourne, based in the Monash Institute of Medical Research and Department of Anatomy and Developmental Biology. He is a structural biologist and reproductive endocrinologist, who has published extensively on reproductive endocrinology, male infertility and the histopathology of the testis. His current interest is the biology of the activins and follistatin in reproductive processes and the applications of the roles played by these proteins in inflammatory and fibrotic diseases as well as genetic causes of male infertility. He is a Fellow of the Royal Australasian College of Physicians, the Australian Academy of Science and the Australian Academy of Technological Sciences and Engineering.