Abstract
Although originally characterised as proteins involved in the control of reproductive function, activins, and to a lesser degree inhibins, are also important regulators of homeostasis in extragonadal tissues. Accordingly, disrupted inhibin/activin expression can have detrimental effects not only on fertility and fecundity but also on the regulation of muscle, fat and bone mass. Indeed, only recently, two complementary mouse models of inhibin designed to lack bioactivity/responsiveness revealed that inhibin A/B deficiency during pregnancy restricts embryo and fetal survival. Conversely, hyper-elevated levels of activin A/B, as are frequently observed in patients with advanced cancers, can not only promote gonadal tumour growth but also cancer cachexia. As such, it is not surprising that inhibin/activin genetic variations or altered circulating levels have been linked to reproductive disorders and cancer. Whilst some of the detrimental health effects associated with disrupted inhibin/activin levels can be attributed to accompanied changes in circulating follicle-stimulating hormone (FSH) levels, there is now abundant evidence that activins, in particular, have fundamental FSH-independent tissue homeostatic roles. Increased understanding of inhibin/activin activity, garnered over several decades, has enabled the development of targeted therapies with applications for both reproductive and extra-gonadal tissues. Inhibin- or activin-targeted technologies have been shown not just to enhance fertility and fecundity but also to reduce disease severity in models of cancer cachexia. Excitingly, these technologies are likely to benefit human medicine and be highly valuable to animal breeding and veterinary programmes.
Structurally related activins and inhibins coordinate the hypothalamic–pituitary–gonadal axis
Inhibins were first postulated a century ago as gonadally derived hormones that could influence pituitary function and follicle-stimulating hormone (FSH) secretion (Mottram & Cramer 1923, McCullagh, 1932) (Fig. 1). However, it was only recently (1992 onwards) that the mechanisms of inhibin-mediated activin antagonism and resulting physiological actions began to be fully uncovered (Fig. 1). Like all members of the large transforming growth factor-β (TGF-β) superfamily, activin A (composed of two βA-subunits, encoded by Inhba/INHBA genes), activin B (βB/βB dimers, encoded by Inhbb/INHBB genes), inhibin A (α/βA dimers, encoded by Inha/INHA and Inhba/INHBA genes, respectively) and inhibin B (α/βB dimers, encoded by Inha/INHA and Inhbb/INHBB genes, respectively) are initially synthesised as large precursor polypeptides that undergo proteolytic maturation prior to activity (Mason et al. 1996). Recognition that activins and inhibins have extensive physiological roles was slowed by their original characterisation as proteins that ‘activate’ or ‘inhibit’ pituitary production of FSH (Ling et al. 1986, Vale et al. 1986). Classically, activins stimulate the production of the FSH β-subunit (Fshb/FSHB) from gonadotroph cells in the anterior pituitary. Structurally related inhibins made by the gonads act in an endocrine manner at the pituitary to suppress FSH β-subunit production. Inhibins constrain both the production and bioactivity of activins, by limiting β-subunit availability during activin formation and by competitively binding to the activin receptors (Lewis et al. 2000, Brule et al. 2021). The opposing activities of these proteins are thought to be integral to the hypothalamic–pituitary–gonadal axis-mediated control of FSH synthesis and therefore, gonadal function. Within the ovary, FSH is required for the commitment of small growing follicles through to the preovulatory stage (McTavish et al. 2007, Bernard et al. 2010). In the testis, FSH promotes germ cell production and androgen synthesis via direct or indirect stimulation of Sertoli cells and Leydig cells, respectively (O'Shaughnessy et al. 2010). Consequently, some of the reproductive changes associated with disruptions to activins and inhibins can be attributed to FSH imbalances.
Timeline for pivotal discoveries in inhibin biology. Outline of the major discoveries in inhibin biology, from the initial indications in 1923 of a gonadally derived factor that could alter pituitary function through to the most recent physiological models that have unveiled new roles for inhibins (Mottram et al. 1923, Setchell et al. 1974, Ling et al. 1985, Miyamoto et al. 1985, River et al. 1985, Robertson et al. 1985, Matzuk et al. 1992, Li et al. 2018, Walton et al. 2022, McCullagh 1932, de Jong et al. 1979, Eddie et al. 1979, Scott et al. 1980, Esch et al. 1987, Forage et al. 1986, Mayo et al. 1986, Mason et al. 1985, Woodruff et al. 1987, Lewis et al. 2000, Brule et al. 2021).
Citation: Journal of Endocrinology 258, 1; 10.1530/JOE-22-0290
In females, inhibin B is produced by granulosa cells of growing ovarian follicles, whereas inhibin A is a product of dominant follicles and, following ovulation, the corpus luteum (Groome et al. 1996). Consequently, inhibin A and inhibin B serum levels fluctuate across the menstrual cycle, with inhibin B levels being highest during the follicular phase and inhibin A levels peaking during the luteal phase (Groome et al. 1996). Intriguingly, males only produce the inhibin B form and not inhibin A, from Sertoli cells in the testis (Illingworth et al. 1996). Inhibin α-subunit production within the somatic cells of the gonads is primarily regulated by FSH, whereas βA/B-subunit transcription is regulated by multiple factors (reviewed in Walton et al. 2011). Accordingly, activin A and activin B are produced by a number of tissues in addition to the gonads, including the kidney (Meunier et al. 1988), bone (Broxmeyer et al. 1988), lungs, liver, heart (Chen & Johnson 1996), adipose tissue (Sjöholm et al. 2006), breast (Di Loreto et al. 1999) and placenta (Petraglia et al. 1987). In contrast, the inhibin α-subunit is almost exclusively expressed within reproductive tissues (gonads, placenta) with the exception of the adrenal glands (Meunier et al. 1988, Voutilainen et al. 1991).
Mechanistically, activin A and activin B mediate activity by docking onto type I and II serine/threonine kinase receptors. Initially, activins bind the activin type II receptors, ActRIIA/B (encoded by ACVR2A/ACVR2B genes), which results in the recruitment and phosphorylation of the activin type I receptors, ALK4 (ACVR1B gene) or ALK7 (ACVR1C gene) (Attisano et al. 1993, Attisano & Wrana 1996). Activin-mediated receptor complex formation typically triggers an intracellular signalling cascade involving the phosphorylation of SMAD2 and SMAD3 transcription factors, their interaction with SMAD4 and translocation into the nucleus (Massagué et al. 2005). Evidence also supports that activin B can also activate the alternate SMAD1/SMAD5 signalling cascade (Canali et al. 2016, Olsen et al. 2020). These activated SMAD complexes interact with SMAD-binding elements in target gene promoters, including the FSHB promoter (Suszko et al. 2005). Inhibin A and inhibin B, in the presence of the co-receptors betaglycan (Tgfbr3/TGFBR3 gene, Lewis et al. 2000) or transforming growth factor receptor-like 3 (TGFBR3L; Tgfbr3l/TGFBR3L gene) (Li et al. 2018, Brule et al. 2021), respectively, form an inert complex with the ActRIIA/B receptors which effectively disables intracellular SMAD activation (Fig. 2A). Activin A and activin B have also been reported to stimulate SMAD-independent signalling pathways involving p38 MAPK, ERK1/2 and JNK (Bao et al. 2005, de Guise et al. 2006). No signalling activity has been identified to date for the inhibin A/B forms.
Schematic outlining the consequences of altered inhibin/activin receptor binding/activation on reproductive and cancer biology. (A) Under physiological conditions, inhibins, in the presence of the co-receptors betaglycan or TGFBR3L, limit activin bioactivity by competitively binding to the activin type II receptors ActRIIA/B to maintain cellular homeostasis. (B) Loss of inhibin bioactivity, or inactivation of inhibin co-receptors betaglycan or TGFBR3L, leads to unopposed activin bioactivity, elevated FSH, altered fertility and embryo and fetal losses. (C) Absence of inhibin production results in pathologically elevated activins which alters cell turnover, drives tumour formation and triggers lethal cachexia wasting. Created with https://www.biorender.com/
Citation: Journal of Endocrinology 258, 1; 10.1530/JOE-22-0290
Roles of activins and inhibins in reproductive physiology
The important roles of inhibin A and inhibin B in coordinating reproductive homeostasis are evident in mouse models in which the activity of these hormones has been disrupted (Fig. 2A and B). It was recently shown that the blockade of inhibin α-subunit maturation effectively inactivates inhibin A/B bioactivity in vivo, as measured by chronically elevated FSH (Walton et al. 2022). Elevated FSH in female mice lacking functional inhibin was associated with heightened ovulation and implantation rates, but this did not translate to more live births, rather there were marked embryo and fetal losses (Walton et al. 2022). Simultaneously, Brulé and colleagues showed that female mice deficient in both the inhibin A/B pituitary co-receptors, betaglycan and TGFBR3L, failed to produce any progeny despite high ovulation rates (Brule et al. 2021). Pregnancy and neonatal loss in mice lacking inhibin A/B bioactivity or responsiveness are likely attributed to altered ovarian hormone milieu, as the ovaries in inhibin-inactivated pregnant dams are significantly enlarged owing to high corpora lutea numbers, triggering elevated serum progesterone levels (Walton et al. 2022).
The reproductive phenotypes in mice lacking inhibin A/B bioactivity or responsiveness are akin to that observed in aged hyper-FSH mice, supporting that pregnancy failure is predominantly attributed to supraphysiological FSH (McTavish et al. 2007). However, there are also likely to be FSH-independent consequences of the loss of inhibin activity on female reproduction, as activins are known to be important for the establishment and maintenance of pregnancy. Indeed, transgenic mice lacking intraovarian βA/B-subunit expression are infertile (Pangas et al. 2007), and unopposed activin activity in follistatin conditional knockout mice results in subfertility owing to defects in uterine receptivity (Fullerton et al. 2017). The βA/B-subunits control ovarian function to varying degrees, as conditional deletion of the βA-subunit results in subfertility, loss of both βA/βB-subunits causes infertility (Pangas et al. 2007). Similarly, genetic inactivation of either SMAD2 or SMAD3 in granulosa cells is redundant for fertility, but the loss of both SMAD2 and SMAD3 leads to severe subfertility and premature ovarian failure (Li et al. 2008).
Within the ovary, activins work in an autocrine/paracrine manner to promote granulosa cell proliferation (Rabinovici et al. 1990, Matzuk et al. 1992) and support oocyte maturation via the upregulation of FSH and luteinising hormone (LH) receptors on the granulosa cells of growing follicles (Nakamura et al. 1993, 1994, Pang & Ge 1999). There are also indications that inhibins promote the production of androgens from theca cells indirectly by antagonising the activities of bone morphogenetic proteins (BMPs) (Glister et al. 2010) and by sensitising the theca cells to LH (Hsueh et al. 1987, Smyth et al. 1994, Wrathall & Knight 1995). Thus, the maintenance of inhibin/activin activity is integral to ovarian-, uterine- and pituitary-mediated control of fertility.
Intriguingly, whilst lack of inhibin A/B bioactivity or responsiveness is associated with infertility/subfertility in female mice, male fertility appears to be preserved, supporting that inhibins, activins and FSH have sex-specific roles (Brule et al. 2021, Walton et al. 2022). Indeed, male mice lacking functional inhibin have normal testis weights and morphology, and daily sperm production is unaltered, despite having a higher FSH tone (Walton et al. 2022). Evidence supports that whilst FSH is redundant for male fertility, it is important for quantitatively normal spermatogenesis, as loss of FSHB expression in male mice is associated with smaller testes and reduced sperm production and motility (Kumar et al. 1997). Independent of FSH, in the mouse, intratesticular activins promote Sertoli cell proliferation during embryogenesis and early post-natal life (Archambeault & Yao 2010) and Sertoli cell differentiation during adolescence (Nicholls et al. 2012) and also regulate Sertoli cell hormone responsiveness (de Winter et al. 1993). Evidence in the mouse suggests that activin A plays a dominant role in testis function relative to activin B and inhibin B, as βB-subunit null mice have normal testes and fertility (Vassalli et al. 1994). However, in men, maintenance of βB-subunit production is important for normal testis function (Meachem et al. 2001, Houston et al. 2022).
Disruptions to activins/inhibin are associated with infertility/subfertility, gonadal tumours and cancer cachexia in mice
Whilst the loss of inhibin A/B bioactivity or responsiveness in the presence of physiological activin A/B results in infertility/subfertility, the complete absence of inhibin A/B production has far more severe consequences for reproductive health (Fig. 2C). Early works revealed that genetic deletion of the inhibin α-subunit in mice (Inha−/− mice) leads to an activin A/B surplus and consequently, the development of ovarian granulosa and testicular Sertoli cell tumours (Matzuk et al. 1992). Serum activin levels increase by as much as 500-fold in adult Inha−/− mice and are associated with lethal cachectic wasting by 12 weeks of age (Matzuk et al. 1992, 1994). Unlike the sex-specific impacts of the loss of inhibin A/B bioactivity on female fertility, complete loss of the inhibin α-subunit results in gonadal tumours and cachectic wasting in both males and females. Thus, maintenance of Inha expression is critical for both sexes in preventing gonadal tumour growth and severe cachexia. Intriguingly, the presence of one intact Inha allele is sufficient to prevent gonadal tumour growth, as heterozygous Inha+/− male and female mice do not develop tumours nor cachexia (Matzuk et al. 1992, 1994), despite age-related disruptions to gonad composition (Itman et al. 2015).
Gonadal tumour formation and severe cachexia in Inha−/− mice are primarily driven by progressively elevated levels of circulating activin A and activin B. In support, ActRII-Fc administration in Inha−/− male or female mice reduces tumour burden, prevents severe weight loss and substantially extends life span (Li et al. 2007). It has also been shown that activins alone, in absence of tumour growth, are sufficient to promote marked wasting of lean and fat mass in mice (Chen et al. 2014, Walton et al. 2019). Disrupted gonadotrophin hormone levels in Inha−/− mice also contribute to tumour growth, as Inha−/− mice lacking FSH (Fshb) or LH β-subunit (Lhb) expression have slowed tumour progression and improved survival rates (Nagaraja et al. 2008, Kumar et al. 1996). Together, these studies highlight the importance of the preservation of inhibin/activin production and bioactivity in the maintenance of reproductive and broader physiological health in mice.
In humans, alterations to inhibins and activins are also associated with reproductive disorders
Human genetic and diagnostic studies corroborate the findings in mice highlighting the importance of inhibins and activins in the maintenance of reproductive health. It has been demonstrated that variations in the INHA, INHBA and INHBB genes are associated with premature ovarian failure (Harris et al. 2005, Chand et al. 2007), ovarian cancer (Tournier et al. 2014) and male infertility (Li et al. 2015, Houston et al. 2022). Only recently, two discrete INHBB gene variations were identified in infertile men (Houston et al. 2022). It was found that an INHBB gene variant c.1079T>C:p.Met360Thr, located in the mature region of the inhibin βB-subunit, resulted in significantly reduced serum activin B levels and altered testis germ cell content in corresponding InhbbM364T/M364T mice. The second INHBB gene variant c.314C>T:p.Thr105Met, which lies within the prodomain of the inhibin βB-subunit, diminished the in vitro production of mature activin B (Houston et al. 2022). Together, this analysis supports that INHBB gene variants that limit activin B production have consequences for testis composition in males. Additional genetic studies in males have identified an association between variants within the INHA promoter region in male infertility patients (Li et al. 2015).
Intriguingly, a recent case study reported the first-ever evidence of inhibin α-subunit null adult humans (Arslan Ates et al. 2022). The INHA genetic disruption (c.208_209delAG:pArg70Glyfs*3) was identified in two adult male siblings who presented with hypospadias, primary hypogonadism, high-normal testicular volume and infertility (Arslan Ates et al. 2022). Indicative of inhibin deficiency, the male siblings also had markedly elevated gonadotropin (FSH and LH) levels and also reduced androgen levels. Testosterone deficiency in these males is likely attributed to impaired inhibin-regulated steroidogenesis in testicular Leydig cells (Wang et al. 2016, Laird et al. 2019). The heterozygous female and male carriers of the same INHA gene variation did not present with any signs of disordered sexual development or infertility. This is indeed an intriguing finding given that male Inha−/− null mice all develop gonadal tumours and succumb to activin-driven cachexia as young adults. In females, it has been shown that INHA and INHBA gene variants that alter inhibin/activin levels are associated with germline ovarian epithelial tumours (Tournier et al. 2014). It has also been demonstrated that an INHA gene variation (c.769G>A:pAla257Thr) identified in a female with premature ovarian failure disrupts inhibin B in vitro bioactivity (Chand et al. 2007). Additional missense variations in the inhibin α-subunit including Ser92Asn, His175Gln and Ala182Asp have been associated with primary ovarian insufficiency and primary and secondary amenorrhoea in women (Dixit et al. 2004, 2006). Together, these studies support that genetic aberrations in inhibins and activins are associated with reproductive disease and signify the importance of these proteins in governing reproductive health.
Clinically, altered inhibin/activin serum concentrations are reliable indicators of diminished ovarian and testis function. In subfertile/infertile men, serum activin B and inhibin B levels are significantly reduced, and FSH is elevated (Pierik et al. 1998, Ludlow et al. 2009). Thus, serum inhibin/activin B concentrations are important biomarkers of testis function in men. In women, serum inhibin A/B precipitously decline at menopause following cessation of ovarian folliculogenesis (Robertson et al. 2008). However, in postmenopausal women with ovarian cancer, circulating total inhibin A/B levels are frequently elevated (Lappohn et al. 1989, Healy et al. 1993, Burger et al. 1996, Lambert-Messerlian et al. 1997). When combined with the CA125 biomarker test, the total inhibin A/B diagnostic has proven to detect up to 95% of ovarian cancers with 95% specificity (Robertson et al. 1999). Polycystic ovarian syndrome (PCOS) patients also appear to have elevated total inhibin A/B serum levels (Tsigkou et al. 2008) and also increased serum follistatin and reduced activin A (Norman et al. 2001). To this day, inhibin A is still included in the quadruple screen test in the second trimester of pregnancy as a reliable indicator of Down’s syndrome (Van Lith et al. 1992, Wald et al. 1996, Haddow et al. 1998). Thus, circulating levels of inhibins and activin are important diagnostic indices of reproductive health status.
Elevated activins are associated with cancers and cancer cachexia
Despite activin A and activin B sharing high sequence, structural and functional homology, activin A has been far better characterised as an underlying pathogenic factor in cancer progression. Elevated activin A expression has been implicated in the pathogenesis of reproductive cancer (Menon et al. 2000, Cobellis et al. 2004, Tournier et al. 2014, Dean et al. 2017, Yi et al. 2019) and various other malignancies in tissues including the breast (Di Loreto et al. 1999, Mylonas et al. 2005, Bashir et al. 2015, Kalli et al. 2019), prostate (Kang et al. 2009, Nomura et al. 2013), pancreas (Lonardo et al. 2011, Zhong et al. 2019, Mancinelli et al. 2021), colon (Wildi et al. 2001, Daitoku et al. 2022) and lung (Seder et al. 2009). Clinically, serum activin concentrations are frequently elevated in patients suffering from lung (Hoda et al. 2016, Paajanen et al. 2019, 2020, Barany et al. 2021), breast (Reis et al. 2002), colon (Loumaye et al. 2015, Bauer et al. 2020, Sartori et al. 2021) and pancreatic cancers (Togashi et al. 2015, Xu et al. 2022).
Whilst the evidence suggests that activins are pro-tumorigenic, activins actually play dichotomous roles in governing cell turnover and metastases. Indeed, it has been shown that activin A can inhibit cell growth in prostate and liver cells (Zauberman et al. 1997), colon carcinoma cells (Jung et al. 2007) and force breast cancer cells into cell cycle arrest (Burdette et al. 2005). Conversely, activin A treatment has been shown to enhance cell growth and proliferation in lung fibroblasts and lung cancer cells (Ohga et al. 1996). Additional studies support a role for activin A and activin B in the regulation of cell migration and metastasis in lung, breast, prostate and oral cancer cells (Tamminen et al. 2015). Together, this suggests that whilst activins are commonly elevated in human cancers, the ability of activins to act in a pro- or anti-tumorigenic manner is tissue specific. Pre-clinical evaluation suggests that activin blockade does not affect tumour size or volume in multiple tumour-bearing mouse models (Zhou et al. 2010, Hatakeyama et al. 2016, Toledo et al. 2016, Nissinen et al. 2018, Zhong et al. 2022), suggesting that activin-targeted technologies may not represent the most effective clinical strategy to reduce tumorigenicity.
Increased circulating concentrations of activin A are also associated with the presence of cachexia in cancer patients and are predictive of patient survival (Loumaye et al. 2015). Supporting studies in mice confirm that supraphysiological levels of activin A or activin B are sufficient to drive the wasting of lean and fat tissues, irrespective of tumour growth (Chen et al. 2014, 2016, Walton et al. 2019). It has also been shown that suppression of activin production (Cadena et al. 2010, Chiu et al. 2013, Latres et al. 2017) or activity promotes muscle hypertrophy (Chen et al. 2015, 2017). Mechanistically, activin-mediated muscle wasting is driven by SMAD2 and SMAD3 which act to transcriptionally regulate E3 ubiquitin ligases including Atrogin-1 (Goodman et al. 2013, Sartori et al. 2009), suppress Akt/mTOR-mediated protein synthesis (Goodman et al. 2013) and decrease the expression of certain myosin isoforms (Loumaye et al. 2022). Whilst activins alone are sufficient to drive wide-spread loss of lean and fat mass, other tumourkines are likely involved in disease progression in patients suffering from cancer cachexia. It has been shown that activin-mediated body wasting is exacerbated by elevated levels of the pro-inflammatory cytokine, interleukin 6 (Chen et al. 2016), which has been implicated in the pathogenesis of cachexia (White et al. 2013, Hetzler et al. 2015, Han et al. 2018, Rupert et al. 2021).
Activin/inhibin-targeted therapies for the treatment of reproductive disorders
Given the ability of inhibins, activins and FSH to control gamete production and embryo development, targeting these factors is an attractive means to modulate fertility. Indeed, recombinant FSH is used to enhance ovulation rates in women undergoing in vitro fertilization (IVF) (Rombauts 2007). Though a reliable means to improve oocyte yields during IVF, in some women, particularly those with PCOS, exogenous gonadotrophins can trigger ovarian hyperstimulation syndrome (OHSS) (Wang & Gemzell 1980, Humaidan et al. 2016). OHSS affects up to 1 in 20 women undergoing IVF and is characterised by (often painful) enlargement of the ovaries owing to a large number of developing follicles, and consequently, heightened serum oestradiol (Papanikolaou et al. 2006, Humaidan et al. 2016). In more severe but rarer OHSS cases, patients can experience symptoms with life-threatening potential including abdominal distension, vomiting and/or diarrhoea, respiratory complications, blood volume insufficiency and thickening, vascular thrombosis and renal failure (Humaidan et al. 2016). A more controlled ovulation induction approach could entail the use of inhibin antagonists to promote more modest increases in FSH and prevent OHSS. Indeed, inhibin immunisation has proven an effective means to enhance female fertility and fecundity in various animal models including sheep (O'Shea et al. 1984, Bindon et al. 1988, Schanbacher 1988), cows, pigs (Brown et al. 1990, Findlay et al. 1993, Jia et al. 2021) and goats (Sasaki et al. 2006). Recent evidence from inhibin inactive/insensitive mice would support that the highest yield of developmentally competent oocytes is achieved with partial inhibin inhibition (Walton et al. 2022). This may be realised using inhibin A- or inhibin B-specific antigens and immunogens or by developing inhibin-targeted ligand traps using the inhibin co-receptors betaglycan or TGFBR3L. Ultimately, inhibin-targeted technologies would have far-reaching benefits in both animal breeding programmes and in allowing more controlled ovulation induction in women undergoing IVF.
Inhibin agonists or mimetics could also prove useful medications for women experiencing natural or iatrogenic menopause. Age-related, surgical or chemically induced loss of ovarian function leads to rapid withdrawal of ovarian hormones including inhibin A/B, in addition to the sex steroids oestradiol and progesterone. Significantly, ovarian inactivation is associated with the onset of numerous, often debilitating, physiological insults in women including disrupted metabolism, accelerated bone and muscle loss, cognitive impairments, urogenital atrophy and sexual dysfunction (Davis et al. 2015). Whilst many of these physiological deficits are attributed to the loss of oestradiol, there is evidence to support that inhibin A/B deficiencies may also contribute. Indeed, precipitously declining levels of inhibin A/B following ovarian inactivation correlate with increased bone turnover (Perrien et al. 2006, Nicks et al. 2010), and supraphysiological levels of inhibin A have been shown to promote bone regeneration in mice (Perrien et al. 2007, 2012). As such, inhibin replacement therapies could be attractive medications for the restoration of bone health in postmenopausal women. Toward this goal, concerted efforts have been made to advance the manufacture of pure and potent inhibin A/B mimetics (Walton et al. 2016, Goney et al. 2020). Unlike steroidal hormone replacement therapies, inhibin A/B treatments are less likely to incur off-target effects owing to tissue-restricted expression of the inhibin co-receptors, betaglycan and TGFBR3L. Further benefits of inhibin replacement therapies on postmenopausal health are yet to be uncovered, but with the new models of inhibin inactivation/insensitivity and inhibin mimetics, uncovering extra-gonadal activities for inhibin A/B is now a feasible goal.
Activin-targeted therapies have been less well explored in the context of reproduction, but there is some evidence to support that activin-ligand traps may prove beneficial for postmenopausal-related muscle and bone loss. Modified versions of the activin A/B propeptides can promote marked muscle hypertrophy in male mice (Chen et al. 2017). Administration of soluble ActRIIB (fused to antibody Fc-chains, sActRIIB-Fc) has been shown to improve muscle mass and function (Cadena et al. 2010, Chiu et al. 2013, Latres et al. 2017) as well as bone mineral density following castration in mice (Chiu et al. 2013, Bialek et al. 2014, Puolakkainen et al. 2017, Puolakkainen et al. 2022). Related ligand trap sActRIIA-Fc has also proven to promote bone growth in mice (Pearsall et al. 2008) and larger primates (Fajardo et al. 2010, Lotinun et al. 2010). Naturally occurring activin A/B antagonist follistatin has also demonstrated to promote marked muscle hypertrophy (Lee & McPherron 2001, Winbanks et al. 2012) and rescue ovariectomy-induced bone-loss in mice (Lodberg et al. 2019, Chan et al. 2021). Whilst encouraging, most of these studies have been conducted in males, and so further studies are needed to uncover the efficacy of these activin-targeted agents in females.
Activin/inhibin-targeted therapies for the treatment of cancer cachexia
Given the underlying role of activin signalling in the pathogenesis of cancer cachexia, numerous strategies targeting the activin-driven SMAD2/SMAD3 pathways have been deployed in pre-clinical models. Some of the most promising developments focused on using soluble activin receptors as ligand traps to bind circulating TGF-β ligands known to promote cachexia. Delivery of the soluble ActRIIB (sActRIIB-Fc) receptor to Inha−/− mice was sufficient to reduce bodyweight loss and extend survival in both male and female mice (Li et al. 2007). Similarly, overexpression of the activin antagonist follistatin in Inha−/− mice reduces body weight loss and extends survival in both male and female mice (Cipriano et al. 2000). Subsequent studies demonstrated that delivery of sActRIIB-Fc to C26 tumour-bearing mice significantly reduced whole-body cachexia and extended life span (Zhou et al. 2010). The preservation of muscle mass in sActRIIB-Fc treated mice appears to be independent of the anabolic process as whole-muscle protein synthesis appeared to be unchanged in C26 tumour-bearing mice receiving treatment (Nissinen et al. 2018). Unfortunately, clinical trials using the sActRIIB-Fc as an intervention for Duchenne muscular dystrophy resulted in off-target effects where patients exhibited side effects including nose bleeding and other vascular complications (Campbell et al. 2017). These side effects were likely a result of the non-specific binding of BMP ligands such as BMP9 and BMP10, which are known regulators of endothelial and vascular cell remodelling (David et al. 2008, Suzuki et al. 2010, Ricard et al. 2012). The recent development of a new heterodimeric Fc-fusion protein consisting of the extracellular domain of the ALK4 receptor and the ActRIIB was shown to promote muscle growth similar to that of the original sActRIIB-Fc (Li et al. 2021). Importantly, this fusion protein did not bind BMP9, reducing the risk of vascular side effects associated with sActRIIB-Fc administration. The ActRIIB:ALK4 heterodimer imparted protective effects in mouse models of disuse atrophy, Duchenne muscular dystrophy and neurodegenerative disease (Li et al. 2021). However, it was not tested in tumour-bearing mice; thus, the therapeutic potential of this novel heterodimer for cachexia remains to be seen.
Other strategies designed to block activin activity in the extracellular space have also been used to reduce cachexia in pre-clinical models. Through the use of recombinant viral vectors and protein delivery, the upregulation of activin A and B propeptides was shown to induce skeletal muscle hypertrophy in mice (Chen et al. 2017, Walton et al. 2019) and block activin-mediated muscle atrophy in various pre-clinical models including the C26 mouse model of cancer cachexia (Chen et al. 2017). These more specific approaches to blocking TGF-β ligands are advantageous as they mitigate the risks of inadvertently inhibiting BMPs or other GDFs as is the case with certain soluble ligand traps.
In addition to extracellular strategies to inhibit activin signalling, efforts have been made to block activin-mediated SMAD2/SMAD3 activity intracellularly. These approaches remove the risk of off-target systemic effects but require suitable transduction methodologies to be able to illicit intracellular efficacy. Adeno-associated viral vector (AAV)-mediated expression of the inhibitory SMAD, SMAD7 is sufficient to block SMAD3 phosphorylation, increase muscle mass, enhance functional capacity and reduce muscle wasting associated with cancer (Winbanks et al. 2016, Maricelli et al. 2018). SMAD7-mediated approaches to combat cachexia may be limited by its negative regulatory role whereby SMAD7 facilitates the recruitment of the Smurf proteins to target TGF-β receptors for degradation (Zhu et al. 1999, Kavsak et al. 2000, Ebisawa et al. 2001). This process may impair BMP signalling which has recently been shown as a critical regulator of skeletal muscle mass and a promising target for treating cancer cachexia (Sartori et al. 2013, 2021, Winbanks et al. 2013). Another specific negative regulator of SMAD2 and SMAD3 mechanisms at the intracellular level is the transmembrane prostate androgen-induced protein (TMEPAI), which is known to specifically bind SMAD2 and SMAD3 but not SMAD1/5/8 (Watanabe et al. 2010), thus not interfering with BMP signalling. It was demonstrated that AAV-mediated overexpression of TMEPAI was sufficient to reduce activin A-mediated muscle atrophy through inhibition of SMAD3 phosphorylation (Hagg et al. 2020). Furthermore, TMEPAI expression was sufficient to reduce muscle wasting in C26 tumour-bearing mice (Hagg et al. 2020) and represents a potential therapeutic strategy for cancer-associated muscle wasting if directed in a skeletal muscle-specific manner.
Concluding remarks
Given the growing body of evidence that activins not only ‘activate’ FSH release to control fertility and fecundity but also have activities in muscle, fat and bone, perhaps it is time to consider renaming these growth factors as ‘SMAD2/SMAD3 activator proteins’ to more accurately reflect their diverse physiological roles. ‘Inhibin’, however, still seems a fitting name, given that most inhibin-mediated bioactivity can be attributed to changes in FSH. Regardless of this, the shared requirement of the β-subunit in inhibin and activin assembly demands that these growth factors are co-examined in physiological contexts. Indeed, this is most evident in the traditional inhibin α-subunit knockout mice, where lethal cachectic wasting occurs not as a direct consequence of inhibin loss, but rather an increase in activin expression. Ultimately, physiological homeostasis is most disrupted when inhibin synthesis or activity is lost or activin production and/or activity is enhanced. An activin production and/or activity bias is disruptive not only to reproductive function, but also has severe consequences for muscle health. Loss of inhibin activity is also detrimental to pregnancy outcomes. The structural and mechanistic discoveries made over decades are now permitting the development of innovative, activin-targeted technologies and inhibin mimetics, which represent an exciting new class of reproductive and muscle therapies.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.
Funding
Project Grant funding (2013284) from the National Health and Medical Research Council (NHMRC) Australia supported this work. This work is also supported by funding from the Faculty of Medicine, University of Queensland.
References
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