New Targets for Old Hormones: Inhibins Clinical Role Revisited

Endocrine Journal 2011, 58 (4), 223-235

Re v i e w New targets for old hormones: inhibins clinical role revisited

Padmanaban S. Suresh1), Thangarasu Rajan2) and Rie Tsutsumi3)

1) Centre for Biomedical Research, Vellore Institute of Technology (VIT) University, Vellore, India 2) Aarupadai Veedu Medical College and Hospital, Puducherry, India 3) University of Tokushima, Institute of Health Bioscience, Department of Public Health and Nutrition, Japan

Abstract. Inhibins are gonadal peptide hormones belonging to the transforming growth factor-β (TGF- β) superfamily that regulate the pituitary follicle stimulating hormone (FSH) secretion by negative feedback mechanisms. It is evident that the understanding of inhibins function in the hypothalamic-pituitary-gonadal axis will provide insights into physiology and pathology of the gonadal function. In recent years, a great deal of attention has been focussed on clinical relevance of measuring circulating inhibins in normal and disease state. The past few years also have witnessed the emergence and discovery of extra pituitary action of inhibins that might provide further insights into the underlying diseases like cancer especially in the reproductive axis and various other new endocrine target organs. In this review after systematic analysis of literature, we discuss briefly the known and recent advances in function of these hormones highlighting also its structure, production and mechanisms of signal transduction. Also this review discusses about the physiological relevance of inhibin association in the normal function to the development of reproductive cancers. Finally, we describe evidence from various emerging studies that inhibins make an important contribution to other physiological functions apart from reproduction which reveals new endocrine target organs of inhibins. The emerging view is inhibin participates in multiple ways to regulate the function in different cell types and still complete repertoire of its actions is under investigation.

Key words: Inhibin, Transforming growth factor, Ovarian cancer, Activins, Betaglycan

Mammalian reproduction is intricate, highly include inhibins and activins, belonging to the trans- ordered and is coordinated by assorted neural, neuro- forming growth factor (TGF-β) super family and fol- endocrine, endocrine, autocrine and paracrine cell-cell listatin (FS), a structurally distinct though function- communication pathways. Many of the pioneering dis- ally related molecule. It has been more than 70 years coveries meticulously demonstrated the roles of hypo- since the identification of negative feedback control of thalamic gonadotropin releasing hormone (GnRH) that pituitary FSH secretion by inhibins. These factors also stimulates pituitary gonadotropins such as follicle stim- were shown to act in autocrine, endocrine or paracrine ulating hormone (FSH) and leutinizing hormone (LH), manner within the reproductive tissues, but the identi- which directly control gonadal functions. Subsequent fication of specific pathways in different cellular con- breakthroughs in the advancement of molecular biol- text along the gonadal axis is still quite unclear and ogy and in understanding signal transduction mecha- should thus be further investigated. The discovery pro- nisms suggested involvement of various cytokines and cess of extra pituitary action of inhibins started almost growth factors at each level of the hypothalamic-pitu- two decades before demonstrating a link between itary-gonadal axis in the paracrine/autocrine control of inhibins and tumour suppressing role in inhibin-α gene different programmed reproductive events. Members (Inha-/-) knockout mice. It was this study that proposed a tumour suppressing role for inhibins when they Received Mar. 23, 2011; Accepted Mar. 29, 2011 as K11E-098 observed initially the development of gonadal and adre- Released online in J-STAGE as advance publication Apr. 9, 2011 nal tumours on knockout of inhibin-α gene (Inha-/-), a Correspondence to: Padmanaban. S. Suresh, Centre for Biomedical subunit of the dimeric inhibins in mice [1]. In accor- Research, Vellore Institute of Technology (VIT) University, dance with the concept of tumour suppressing role of Vellore, India. E-mail: [email protected] /[email protected] inhibins, many studies reported in different tumours Conflict of interest: None such as ovarian, breast and endometrial in human stud- ©The Japan Endocrine Society 224 Suresh et al. ies and current literature point out that inhibins might tion of function of the pituitary gland was further rein- act as tumour suppressors [2]. Beyond the legitimate forced [10]. Apart from the initial studies, that paved surprise that these observations generated about the way for the discovery of inhibins in male reproductive new role of inhibins, they raised many important bio- system, the presence of inhibins like activity was also logical questions and were approached by many clini- observed in the ovarian follicular fluid which was first cal endocrinologists, reproductive and molecular biol- postulated by Sherman and Korenman, (1975) [11]. To ogists independently. Recent, clinical studies where in support the presence of inhibins in follicular fluid, stud- the correlation of serum circulating levels of inhibin in ies were conducted by administration of steroid free various diseases like rheumatoid arthritis, hypogonad- bovine follicular fluid (bFF) to castrated rats which otropic hypogonadism, constitutional pubertic delay, suppressed serum FSH levels [12]. Later, isolation and preeclampsia, ovarian cancer and epithelial ovarian characterization of inhibins was made possible with the cancer has further strengthened its clinical relevance development of an improved bioassay based on mea- [3-7]. Their identification in several tissues has also surement of FSH production by rat pituitary cells in suggested that they have diverse physiological roles vitro [13-16]. Subsequent breakthroughs in the molec- apart from hypothalamic-pituitary-gonadal axis. Given ular biology and its applications, cloning of the gene the important clinical applications of inhibins measure- encoding inhibin was made possible following char- ment in various disease states and also identification acterization and purification from the bovine follicu- of its presence in various different tissues, a greater lar fluid [17-19]. Following, large scale productions of understanding of its physiological role is undeniably recombinant DNA-derived inhibin, great strides were needed. In this review considering the recent advances made in elucidating its role and other functions [20]. in understanding, we highlight the production, struc- Historical retrospective of inhibins research shows that ture-function, signal transduction, bioavailability and many studies were required to reach the current under- action of inhibins in gonads and other peripheral tis- standing of its structure and function and still the com- sues (new endocrine roles). plete repertoire of inhibins role and its signal transduction mechanisms are unknown and opens opportunities Historical Background of Inhibins for future research. Discovery Structure of Inhibins The perception of non-steroidal substances produced from the gonads regulating the function of anterior The mature heterodimeric bioactive inhibin mole- pituitary gland was not new and known almost more cules consist of a α-subunit (~20kDa) and two highly than 75 years ago. The destruction of the seminifer- related β-subunits (~14kDa). Both the subunits are ous tubules by X- irradiation of the testis also caused linked by disulfide bonds to form either inhibin A (α, hypertrophy of the anterior pituitary gland and hyper- βA) or inhibin B (α, βB) isoforms. In contrast, homo- plasia in many cells of the pituitary in experimental or hetero dimers of β subunits form the other members rats [8]. This unexpected observation led to prediction of TGF-β activin A, activin B or activin AB (Fig. 1). of the association and the functional feedback regula- These three subunits (α, βA, βB) are products of sepa- tion between the gonads and the pituitary gland for the rate genes located on chromosomes 2 (α and βB sub- first time. If gonads functioned to regulate the pituitary unit) and 7 (βA subunit) [6]. There is 23-27% amino gland, identification of the contributing substance(s) acid homology between the α subunit and β subunit from the gonads was the next challenge in the field of and 64% homology between the β subunits [6]. Each inhibins discovery process. It was only after a decade subunit is initially assembled as a large pre-pro-protein later, McCullagh in 1932 demonstrated that a water sol- which possess a N-terminal signal peptide (a pro-re- uble extract from bull testis prevented post castration gion needed for the accurate folding and dimerization hypertrophy of the rat pituitary gland and he named it of the subunits and which targets the polypeptide to as ‘inhibin’ for this proteinaceous substance [9]. In the the endoplasmic reticulum) and the mature C-terminal 1970’s, with the advent of methods for portrayal and domain. Proteolytic partial cleavage of pro-region measurement of circulating pituitary gonadotropins makes it biologically active. Partial processing of pro- (FSH & LH), the concept of inhibins role in the regula- forms and glycosylation pattern on C-terminus of the Clinical roles of inhibin 225

Fig. 1 Schematic of the precursors of the inhibin α, βA and βB subunits and their dimerization pattern. Details on structure of the precursors and assembly are provided in the text. The mature proteins are indicted by the shaded areas. The formation of disulfide bonds between the two mature subunits are shown by S-S. aa- amino acids. subunit will give rise to different isoforms of inhibin Localization and Biosynthesis of and have been reported in bovine and human plasma Inhibin in Gonads [21-23]. Non bioactive forms of the α-subunit exist as pro-αN, pro-αC and further processed. Though these A. Inhibin in male gonads subunits lack fully functional inhibin activity in the Inhibin subunits are present in several cell types of pituitary FSH release bioassay, it is not yet clear of any testis including Leydig and Sertoli cells and the rela- biological effect on the reproductive system. For both tive expression of the subunits (α, βA and βB) appear isoforms, the glycosylation sites are at either Asn (268) to change throughout the sexual development in rats or Asn (302) in α-subunit to produce 31 and 34 kDa iso- [31, 32]. In rats, Sertoli cells are presumed to be the forms. Makanji et al. [24] observed a decrease in bio- chief source of circulating inhibin and to express α, βB activity on glycosylation of Asn (302) of the α-subunit but to a lesser extent βA subunit [32]. Inhibin βB sub- of inhibin A and B. This effect was explained by the unit mRNA expression was also found to be intense decreased affinity of isoform inhibin A to betaglycan in germ cells from spermatogonia to round sperma- [24]. The pattern of post translational modifications tids other than Sertoli cells in human adult testis [33]. that regulates the bioactivity of inhibins from its vari- Circulating serum levels of inhibin B are strongly and ous functions in both normal and disease condition is positively correlated with testicular volume and sperm still to be understood. The assembly of inhibin A and B counts [34]. Apart from the regulation of pituitary and other TGF-β super family ligands and its secretion FSH secretion by negative feedback loop, inhibins also are dependent upon non-covalent interactions between act as potential paracrine and autocrine regulators of their pro-and mature domains [25]. Conservation of Leydig and Sertoli cell proliferation, differentiation these residues in all TGF-β super family ligands sug- and steroidogenesis in vitro [34]. These autocrine and gests a common biosynthetic pathway governing the paracrine roles have not yet been determined in vivo. dimer assembly [25]. Aberrant expression in the levels In contrast, to the male gonads, the autocrine and para- of subunits and its receptors has been reported in vari- crine roles of inhibins are much evident within the ous disease conditions [26-30]. This makes critical to reproductive tissues of the females [35]. understand the subunits synthesis, assembly, secretion and functions of inhibins under a variety of physiologi- B. Inhibins in female gonads cal and pathophysiological conditions. Differential regulation in production of different isoforms of inhibin in ovaries and other reproductive tissues such as endometrium and placenta makes 226 Suresh et al. female reproductive system an unique model to study clearly established and rigorous testing is needed to the diverse role of inhibins [35-37]. Ovarian inhibin is evaluate its identity. Several studies report the evi- primarily expressed in follicles and its presence in the dence in support for the presence of inhibin receptors corpus luteum (CL) is species specific [38]. The pri- such as the identification of inhibin specific binding mate CL is unique, in that along with progesterone (P4) sites in normal and tumour tissues [50, 51]. Inhibin biosynthesis, it also synthesizes substantial amounts of specific membrane associated binding proteins were estradiol (E2) and inhibin A isoform. The fact that CL purified from gonadal tumours in inhibin-α knockout is the major source of inhibin during the luteal phase mice [50]. It was proposed that down stream signaling was confirmed by the observation of rapid decrease in components of inhibin receptor might relay serine-thre- immunoreactive inhibin levels following lutectomy in onine kinase activity. However attempts to clone addi- cynomolgus monkeys and specific inhibin A levels in tional members of the receptor serine-threonine kinase bonnet monkeys [39,40]. The secretion of inhibin A family have failed [49]. Since attempts to character- in CL appears to be regulated by LH, since inhibition ize the independent signaling components of inhibins of pituitary LH secretion by treatment with gonado- failed, it was proposed that inhibin could act by domi- tropin releasing hormone receptor (GnRH-R) antago- nant negative regulation of the activin signal transduc- nist caused a rapid decline in circulating inhibin and tion pathways [49]. The βA subunit of inhibin A can inhibin-α subunit mRNA levels [40-44]. In primates, bind to activin type II receptors with low affinity and circulating levels of inhibin A and P4 manifested con- binding blocks activin interaction with its receptor that cordant changes such that both exhibited rise and fall uncouples activin signal transduction pathways (Fig. during the luteal phase and both decreased following 2). The affinity of inhibin A for activin type II receptor lutectomy which confirms the source of inhibin secre- isoforms (ActRII A and ActRII B) has been reported to tion during the luteal phase [39, 40, 45]. Inhibin A bio- be 2-10 fold lower than that of activin A [52]. This sug- synthesis in CL appears to be present only in higher gests that inhibin must be present in excess to antago- primates since expression of inhibin-α subunit has nize the activin actions. It has also been reported in been reported to be turned off following luteinization some systems where inhibin A can antagonize activin in rodent and bovine species [46-48]. A actions at equimolar or even lower concentrations [52]. Some activin responsive cell lines are insensi- Mechanisms of Inhibin Signal tive to inhibins even at higher levels which also sug- Transduction gest that competitive binding alone cannot account for antagonism [52-54]. Several studies have identified The independent downstream signaling cascade of non-overlapping binding sites for activin A and inhibin inhibins is poorly understood and the efforts to under- A in various tissues which suggests for the existence of stand is greatly enhanced by discoveries in related to inhibin specific receptors [49]. There are two candidate TGF-β superfamily members, and our current knowl- inhibin receptor proteins studied so far and they are edge based on several studies in this regard is discussed Betaglycan and inhibin binding protein/p120 (INHBP/ here. In comparison to the available knowledge on the P120) [49]. Betaglycan was originally characterized as cellular actions of activins, there is less succession of the TGF-β type III receptor [52]. It was shown to bind knowledge in studying inhibin actions and the reasons TGF-β3 with high affinity and to be necessary for high are due to following: 1) Inhibins are mammalian con- affinity association with TGF-β2 with its type II recep- strained hormones and are difficult to study in other tor. Betaglycan surprisingly was also found to bind model systems [49], 2) There exists enormous difficul- inhibin A with high affinity suggesting it as a potential ties in purification of native inhibin and also expres- receptor [52, 55]. Betaglycan possess distinct inhibin sion and purification of recombinant inhibin [49], 3) and TGF-β binding sites and suggest that betaglycan, There are constrained target sites for inhibin in cells may function as a co-receptor for these ligands [56]. of reproductive and non-reproductive tissues, [49] 4) Disruption of the betaglycan binding by simultaneous There is lack of a clearly acknowledged receptor and substitution of Thr (111), Ser (112), Tyr (120) to Ala target genes modulated by inhibin have not been iden- yields an inhibin A variant that is unable to mediate tified [49]. If in fact a signal is generated in response its action i.e., unable to suppress activin-induced FSH to inhibin, the identity of such a messenger should be release by rat pituitary cells in vitro [24]. The other Clinical roles of inhibin 227

Fig. 2 Inhibin signal transduction Activin binds to the serine threonine kinase type I and type II receptors present on the cell surface. Type II receptor gets phosphorylated upon binding of the ligand and activates the type I receptor. Type I receptor phosphorylates downstream R-SMADs. Phosphorylation of R-SMAD associates with the Co-SMAD (SMAD4) and enters the nucleus to initiate the transcription of target genes along with other cofactors. Inhibin binds to the activin type II receptors and block recruitment of type I receptors and after steps of activin signaling. candidate receptor INHBP/p120 is a large transmem- fluid or recombinant (rh) inhibin A’s preparations eluci- brane protein with a short, kinase deficient cytoplas- dated the role of inhibin in the control of pituitary func- mic tail, and was purified from bovine pituitary mem- tion. Exogenous administration of inhibin has been brane extracts by its affinity for inhibin A [52]. Unlike studied in a variety of animal model systems for exam- betaglycan, INHBP forms a complex with the activin ple; administration of rh inhibin A to the rats of both type I receptor, ALK4 in a ligand independent man- sexes, caused dose and time-related decrease in plasma ner. INHBP does not disrupt activin signal transduc- FSH, but not LH levels [59]. There was lowered cir- tion in the absence of Inhibin. Activin stimulated gene culating FSH levels and reproductive defects in inhibin transcription was blocked by INHBP in the presence α-subunit knockout mice when inhibin A was exog- of Inhibin B, but not Inhibin A, which suggests that enously regulated in the liver tissue [60]. Numerous INHBP may function as an inhibin B specific receptor studies in different animal models concluded that sys- [52, 57, 58]. temic administration of inhibin suppresses pituitary FSH secretion, while immunoneutralization of inhibin Pituitary Actions of Inhibins leads to increase in serum FSH levels [61]. When the bioactivities of highly purified preparations of recom- Although the inhibin-α subunit mRNA and protein binant human 31-kDa inhibin A and B were deter- are expressed in the anterior pituitary, the major endo- mined in rat pituitary cells in vitro, and in ovariecto- crine feedback action upon pituitary FSH secretion mized adult rats in vivo based on suppression of plasma seems to be from inhibin of gonadal source [35] (Fig. FSH, it was found inhibin B has more potent activity 3A). As shown in Fig. 3B, the interaction of inhibin- than the other [62]. Gonadotrophs are the major pitu- α with various proteins as analyzed by STRING net itary targets of inhibin and the majority of these cells work indicates their functional complexity. Numerous express the inhibin co-receptor betaglycan [55, 58, studies using either partially purified ovarian follicular 63]. The presence of receptors for inhibin also has 228 Suresh et al.

Fig. 3A The Hypothalamic-pituitary-ovarian axis Inhibins secreted from the ovaries negatively regulate the pituitary FSH secretion whose secretion is also controlled by hypothalamic GnRH. 3B Protein interactome of inhibin alpha analyzed by STRING 8 network 104 Protein association network in STRING version 8.0 centred at the query protein inhibin alpha. Different line colours connecting different proteins represent the type of evidence for the association. The factors such as CREB, SF-1 and GATA-4 has been documented by evidence of association; FSH-follicle stimulating hormone; FST-follistatin; ACVR2A- activin receptor 2A; TGF-transforming growth factor; AMH- anti mullerian hormone been identified in the thecal and granulosa cells of the active inhibin was reported in the late follicular phase ovary [63-65]. Devoid of apparent recognition of sig- and reaching a peak with the LH surge in the men- naling effectors downstream to inhibin signaling, it is strual cycle [45]. The observed highest levels during antagonism of activins action by inhibins that modu- the luteal phase positively correlated with high P4 lev- late the functions of gonadotrophs in its FSH secretion els in primate menstrual cycle. Studies demonstrating and FSH-β expression [66, 67]. Inhibins also modulate the ability of human chorionic gonadotropin (hCG) to gonadotroph sensitivity to GnRH receptors [68]. prevent the luteal regression and premenstrual decline of serum inhibin levels during the late luteal phase and Inhibins and Reproductive Physiology following GnRH antagonist administration [69] confirmed CL being the source of inhibin during the luteal There is availability of substantial data elucidating phase. Studies involving lutectomy in monkeys and the role of the inhibins in females compared to males. other studies in human further confirmed the source This is because of the success to conduct in vitro cul- of circulating inhibin during the luteal phase in the ture studies in granulosa cells and failure to achieve reproductive cycle [39, 40, 70]. Subsequent develop- similar studies in seminiferous epithelium. Initially ment of sensitive assays and measurement using these quantitation of inhibin was done using radioimmuno- assays showed similar pattern of secretion of inhibin assay in many studies which also measured products A. In contrast, to the circulating profile of inhibin A, of the α-subunit. This cross reactivity resulted in an inhibin B levels are highest during early to mid follicu- inability to distinguish between inhibin A, inhibin B lar phase and decline in the late follicular phase [71]. and α-subunit proteins that were present in the circula- Inhibin B appears to be absent in the primate CL [71]. tion. Using this assay, a modest increase in immunore- The secretory patterns of inhibin and other hormones Clinical roles of inhibin 229 that regulate reproduction are different in humans and Inhibins in Clinical Reproductive rodents. For instance, in contrast to human data, LH Medicine causes a rapid decrease in inhibin production (serum and mRNA levels) in the rat. The CL of the rat does There are diverse areas of investigation of inhibins not express inhibin subunit mRNA’s and does not pro- in obstetrics and gynaecology. Most of clinical stud- duce inhibin. Both inhibin A and inhibin B have the ies on inhibins are mainly focussed on reproductive tis- capacity to suppress FSH secretion however, the rela- sues such as the ovary, placenta and the testis. Inhibin tive potency of inhibin A and inhibin B had been dif- can be used as a clinical marker for prenatal screening ficult to assess due to use of different bioassays and of Down’s syndrome [76, 77]. Measurement of cir- immunoassays employed by various groups. However, culating levels of inhibins also aids in investigation of recently Makanji et al., 2009, reported that inhibin B is reproductive ageing and assessment of in vitro fertil- a more potent suppressor of rat FSH both under in vivo ization [78, 79]. It has been reported that inhibin is and in vitro conditions [62]. As the levels of inhibin useful as a clinical marker for granulosa cell tumour A and E2 rise in similar courses, it is difficult to delin- and mucinous carcinoma of the ovary [80]. It is a eate the specific role for each in the control of the FSH good marker to assess CL function upon P4 supplemen- in the late follicular phase. The differential patterns tation during luteal insufficiency. The specific assays of inhibin A and inhibin B reflect the localization and for both the isoforms of inhibin also suggested a dif- production of these two proteins in the various stages ferential role that inhibin B may be a useful marker of follicular development [72]. The preantral follicle for follicular development and that inhibin A may indi- shows evidence of βB production with no α-subunit cate functionality of CL. Recently single nucleotide being detected suggesting that the dimeric product is polymorphisms (SNPs) of inhibin A gene and its asso- activin B. FSH stimulates α-subunit production which ciation of premature ovarian failure have gained a lot together with βB subunit is responsible for inhibin B of attention in clinical research related to reproductive production when the cohort of follicles proceed towards disorders [81-83]. Activin A and inhibin A measure- ovulation. The dominant follicle produces βA resulting ment throughout pregnancy may have clinical useful- in increased inhibin A production during the later part ness in terms of diagnosing many gestational diseases of the follicular phase to peak at the mid cycle. This (37) Both inhibin A and activin A serve as markers for in turn decreases FSH leading to the suppression of the assessment of perimenopausal changes, trophoblast βA expression in small follicles. LH surge is associated viability, tumours and diagnosis of hypertensive distur- with decrease in the expression of the inhibin subunits bances of gestation [84]. and after formation of CL, expression of α and βA subunits are maintained in primates. Inhibin exerts stimu- Inhibins and Its Role in Tumourigenesis latory effects on folliculogenesis and may control follicular growth, development, atresia, oocyte maturation The inhibin field research in tumour biology is con- and spermatogenesis [73, 74]. Mice bearing a targeted founded by the available data that inhibin-α acts as a deletion of the α-subunit gene generated by homologous tumour suppressor in adrenal glands and ovaries of recombination in mouse embryonic stem cells develop mice however elevated in circulating concentrations in normally, but all of them eventually develop mixed or women with ovarian cancer [2, 85]. Studies on knock- incompletely differentiated gonadal stromal tumors [1]. out mice for inhibin-α subunit gene showed aggres- This suggested that inhibin is a critical negative regula- sive Sertoli/granulosa cell tumours in the gonads and tor of gonadal stromal cell proliferation, and it is anti- adrenals of both sexes in mice [1]. These reports sug- tumorigenic. The human placenta is a major source of gested that inhibin-α act as putative tumour suppres- plasma inhibin-like activity and placental cells in cul- sor in mice gonads and adrenal gland. But in humans, ture secrete these proteins [75]. The α-subunit and its granulosa cell tumours are initiated when the circulat- mRNA are found in the cytotrophoblast region of the ing levels of inhibin are high, and suggest that gran- villi, whereas βB is expressed in the syncytial layer. The ulosa cell tumours in human may be associated with βA protein is expressed in both inner and outer layers. loss in the responsiveness rather than loss of expres- The expressions of all these subunits are increased dur- sion [86]. In support of this hypothesis, it was found ing the progression of pregnancy [75]. that almost 53% of granulosa cell tumours exhibited 230 Suresh et al. decreased receptor expression (TGFβ3) when com- Table 1 Association of Inhibins in various disorders pared to normal premenopausal ovary and ovarian and Disease References testicular malignant germ cell tumours exhibited posi- 1 Rheumatoid arthritis & systemic lupus erythematosus [3] tive staining for inhibin/activin subunits [87, 88]. In 2 Hypogonadotropic hypogonadism [4] humans, it is reported that endogenous inhibins con- 3 Preeclampsia [5] 4 Ovarian granulosa cell tumours [87] trol the granulosa cell tumour migration and inva- 5 Down syndrome [76] sion by acting through TGFβ3 receptor [87]. Using 6 Ovarian cancers (mucinous and sex cord stromal) [80] Xenograft model of epithelial ovarian cancer cell lines, 7 Premature ovarian failure [81-83] it was demonstrated that on loss of inhibin responsive- 8 Gonadal malignant germ cell tumours [88] ness there was more aggressive tumour phenotype in 9 Breast cancer [26] the cell lines both in vitro and in vivo condition [89]. In 10 Prostate cancer [91, 92] accordance with this, when TGFβR3 is over expressed 11 Endometrial cancer [29] in ovarian cancer epithelial cells, it blocked cell migration, whereas inhibin-α subunit silencing enhanced both invasion and migration [90]. In contrast to the in association with increased circulating FSH levels inhibin association in ovarian cancer, the expression [95]. Also it was correlated that decline in gonadal of inhibin-α have been reported to be high in the late- inhibins strongly associate with increase in bone for- stage and metastatic prostatic cancers [91, 92]. These mation and resorption markers across the menopause studies demonstrated a pro-tumourigeneic and promet- transition [95]. Circulating concentrations of inhibins astatic function of inhibin-α associated with androgen predict lumbar bone mass in perimenopausal women independent stage of late and metastatic prostatic dis- regardless of changes in sex steroids or FSH levels ease. Additionally it was shown that the expression of [95]. Supporting this hypothesis, in vitro studies in inhibin-α subunit could serve as a predictive factor for murine bone marrow cell cultures demonstrated a direct the prognosis of prostate cancer [92]. It is reported that effect of inhibin A and inhibin B upon osteoblast and there is a differential expression of inhibin-α in ductal osteoclast differentiation [96]. It was also shown that carcinoma in situ (DCIS) which suggest a tumour sup- continuous exposure of mice to inhibin A in vivo pro- pressor role for inhibin-α in breast tissue [26]. Analysis tected the bone loss after gonadectomy and regulated of inhibin/activin- alpha, -beta A and –beta B subunits bone quality [95]. It was demonstrated that inhibins in 302 endometrial cancer tissue samples by immuno- suppressed the recruitment of cells into the osteoblastic histochemical analysis revealed a significant correla- lineage as well as osteoblastic differentiation into bone tion with histological grading, surgical staging, lymph nodules [96]. The demonstration of suppressive role node status and diabetes in patients with endometrial on multi-nucleated tartrate-resistant acid phosphatase cancer [29]. In vitro studies in endometrial Ishikawa positive (TRAP+) cells, indicator of osteoclastogene- cell line demonstrated a stimulating role for interferon sis further supports the bone remodelling role of inhib- beta1a upon inhibin-α expression which opens new ins [95]. The mechanism of all these actions was often therapeutic strategies for the treatment of endometrial correlated with blocking action on bone morphoge- cancer [93]. The differential expression of inhibin beta netic proteins effect on osteoblastogenesis and osteo- A and beta B subunits in normal and malignant glandu- clastogenesis. Subsequently, the confirmation of these lar epithelial cells of the human uterine cervix suggest actions in human cells provided further evidence that an important role for inhibin beta units in cervical can- changes in ovarian inhibins might directly influence cer [94]. Therefore, all these studies suggest inhibin/ bone turnover via changes in cell differentiation [97]. activin subunits might serve as useful prognostic mark- Clinical studies in peri-menopausal women that mea- ers in tumourigenesis and the related studies are indi- sured serum circulating levels of steroids, inhibins and cated in (Table 1). FSH confirmed that decrease in inhibin levels across the menopausal transition is associated with increasing Inhibin and the Regulation of Bone Mass bone turnover as well as with bone mass regardless of changes in steroids or FSH levels [95, 97, 98] (Fig. 4). Perimenopausal changes in bone i.e., increased bone Together, these data suggest that inhibins regulate bone turn over and bone mass have been reported in women mass by endocrine regulation of bone metabolism and Clinical roles of inhibin 231

covered in the 1930s. Emerging and unexpected new roles from inhibin accentuate the significance to study the regulatory processes involved in its production and action on that particular tissues. Advanced details about the molecular mechanisms involved in assembly, signal transduction, differential regulation of the two iso- Fig. 4 Changes in hormones dynamics in association with bone health during premenopausal, peri-menopausal forms and biosynthesis of inhibins in normal and dis- and post menopausal state (See text for details) ease state is yet to be resolved. These vitally important questions are currently being addressed in many labo- ratories as evident in the recent years by rising amount hypothalamic-pituitary-gonadal axis can be extended of information available in the literature. By technol- to include its effects on skeleton. ogy and modern sophisticated research approach, it should become possible to address all the important Inhibins in Digestive System questions on inhibin’s biology and its new endocrine roles in the coming new years. This will further help Gastro-intestinal and pancreatic endocrine cells pro- in our understanding to combat against the issues of duce several hormones and/or amines and belong to dif- fertility, reproductive cancers and bone homeostasis fuse enteric endocrine system. Additionally to secre- problems (osteoporosis). It could also envisage novel tion of hormones, this group of cells secrete growth therapeutic strategies in the diagnosis and treatment of factors and express its receptors as evident from many related tumours. In summary, the field of inhibin biol- studies [99-101]. It has been demonstrated that human ogy and particularly its association with clinical disor- endocrine cells of the gut express activin A and pancre- ders remain a fruitful area of research enquiry while atic islet cells in rats express beta A and activin [102, striving for therapeutics. 103]. The findings of differential expression of activin/ inhibin subunits among different types of human gas- Acknowledgements trointestinal and pancreatic endocrine cells and related tumours suggest a modulatory role for these factors in PSS would like to thank Dr. Medhamurthy, MRDG biological functions of these cells in the gut. Dept, Indian Institute of Science, Bangalore for intro- ducing this unique hormone during his research work. Concluding Remarks We would like to thank management of VIT for provid- ing help and support. We apologize to many scientists Albeit the identification of true inhibin receptor is whose work was not given credit due to space limita- still to be resolved, several lines of evidence support the tions and in some cases reviews have been cited at the direct role of inhibins in many physiological functions expense of the original work. apart from originally proposed when they were dis-

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