Follistatin/activin-binding protein.

Follistatin, or follicle-stimulating hormone (FSH) suppressing protein, is a protein isolated from side fractions in the purification of inhibin and activin, which shares with inhibin the property of suppressing the secretion of FSH from the pituitary gonadotroph. This protein is structurally distinct from inhibin. Recent observations that follistatin is an activin-binding protein suggest that it may have a much wider biologic role, given the variety of systems in which activin has been shown to act.     

Follistatin binds to both activin and inhibin through the common subunit

Inhibin, activin, and follistatin are three families of polypeptides originally isolated and characterized from ovarian follicular fluid based on their modulation of FSH release from pituitary cell culture. In addition to their effects on FSH synthesis and secretion, inhibin and activin have other biological functions. By contrast, the physiological significance of follistatin was obscure, until it was discovered that follistatin is a binding protein to activin. Since activin binds to follistatin, it is imperative to determine the nature of the activin/follistatin binding complex. Moreover, because inhibin contains a beta-subunit derived from activin, it is important to determine whether inhibin will also bind follistatin. Using a double-ligand blotting technique, we have determined that activin-A has two binding sites for follistatin, whereas inhibin-A has only one binding site for follistatin. Therefore, these results suggest that follistatin binds to both activin and inhibin through the common beta-subunit.     

Regulation of follicle-stimulating hormone secretion by the interactions of activin-A,dexamethasone and testosterone in anterior pituitary cell cultures of male rats

This study was designed to evaluate the effects of glucocorticoids and gonadal steroids on the expression of inhibin/activin subunits and follistatin of the anterior pituitary and test the hypothesis that resulting changes in the local activin/inhibin/follistatin tone contribute to steroid effects on follicle stimulating hormone (FSH) production from gonadotropes. In primary cell cultures of male rat anterior pituitaries, dexamethasone (DEX) or testosterone (T) stimulated FSH secretion and FSHbeta mRNA and their effects were additive with activin-A. Follistatin (FS288) and inhibin-A antagonized the rise in FSH secretion both in the absence and presence of exogenous activin-A. Despite the similarity in their action on FSH production, DEX and T had opposite effects on follistatin mRNA levels. Follistatin mRNA levels of cultured rat anterior pituitary cells were elevated upon the addition of DEX but attenuated by T. On the other hand, both DEX and T suppressed inhibin/activin betaB mRNA levels while only DEX affected betaA mRNA. In these cells, activin-A stimulated follistatin and inhibin/activin betaB mRNA levels but had no effect on betaA. Together, DEX and activin-A caused a further increase in follistatin mRNA levels while T attenuated the effect of activin-A alone. Both steroids attenuated the effect of activin-A on betaB mRNA accumulation. These results support the possibility that DEX and T, possibly acting on different subsets of anterior pituitary cells, use distinct mechanisms to modify the local activin/inhibin/follistatin circuitry and thereby upregulate FSH production from the anterior pituitary gonadotropes.     

Effect of adenovirus-mediated overexpression of follistatin and extracellular domain of activin receptor type II on gonadotropin secretion in vitro and in vivo

Activins are dimeric proteins that stimulate the synthesis and secretion of pituitary FSH by interacting with two classes of receptors, type I and type II, to initiate their intracellular signaling cascade. The extracellular domain of type II activin receptor (ActRII-ECD) contains all structural determinants sufficient for high affinity ligand binding. A soluble recombinant ActRII-ECD has been reported to attenuate FSH secretion from cultured rat anterior pituitary cells in response to exogenous activin A or endogenous activin B. Follistatin is a binding protein that acts as an extracellular factor to bind and inactivate activin. We constructed adenoviral vectors able to mediate expression of follistatin 288 (AdexCAFS288) and ActRII-ECD (AdexCAECD) and tested their biological activities both in vitro and in vivo. The data show that adenovirus-mediated overexpression of either ActRII-ECD or follistatin was able to attenuate FSH secretion by cultured rat anterior pituitary cells. However, AdexCAFS288 overexpression of follistatin was more effective than adenovirus-mediated overexpression of ActRII-ECD. In vivo, a single ip injection of AdexCAFS288 induced the expression of high levels of follistatin and resulted in the suppression of serum FSH levels in castrated male rats for up to 12 d postinjection. Infection with AdexCAFS288 had no effect on LH secretion in vitro or in vivo, demonstrating its selectivity. In conclusion, the results demonstrate the effectiveness of adenovirus-mediated overexpression of follistatin and ActRII-ECD to regulate FSH secretion and the potential of using this strategy as a tool to further define the critical role of activin/inhibin/follistatin circuitry in the modulation of the reproductive system.     

Activin-binding proteins in human serum and follicular fluid.

Binding proteins that transport and/or modify the biological action of peptide hormones and growth factors have been identified for an increasing number of endocrinologically important substances. Since these binding proteins can mask epitopes critical for recognition in immunoassays and can neutralize the bioactivity of their targets, elucidation of hormonal physiology can be intricately tied to analysis of binding protein structure and function. Therefore, we investigated whether circulating activin- and inhibin-binding proteins exist in human serum by incubating purified recombinant human 125I-activin with serum samples. After gel permeation chromatography, radioactive activin was identified in three peaks, a high molecular wt (mol wt) binding protein peak (230 kDa), a lower mol wt binding protein peak (60 kDa), and free activin (22.5 kDa). Bound activin was displaced from the lower mol wt binding protein with either activin or inhibin, but was not displaced from the high mol wt peak with a 10-fold greater concentration of activin. Since an activin-binding protein, follistatin, has been identified in ovarian and pituitary extracts, these same analytical techniques were applied to analysis of human follicular fluid as well. A large, 60 kDa binding protein peak eluting in a position similar to the lower mol wt peak in serum was observed, consistent with this protein being follistatin. These results demonstrate the presence of at least two activin-binding proteins, distinguishable by size, in human serum that may interfere with attempts to assay activin levels in circulation without prior extraction, and may also be involved in regulating the biological actions of activin.     

In vitro secretion of FSH by cultured clinically nonfunctioning and gonadotroph pituitary adenomas is directly correlated with locally produced levels of activin A

OBJECTIVE: Expression of mRNAs encoding activin and its antagonists inhibin and follistatin has been described in human pituitary adenomas, including clinically nonfunctioning adenomas (NFAs) and gonadotroph adenomas (Gn-omas). Since many of the NFAs and Gn-omas secrete FSH in vitro, we hypothesized that locally produced activin may stimulate secretion of FSH in these pituitary adenomas. PATIENTS AND METHODS: Pituitary adenoma tissue was obtained from 38 patients diagnosed preoperatively as having NFAs (n = 17), Gn-omas (n = 5), prolactinomas (n = 6) or growth hormone (GH)-producing adenomas (n = 10). Actual protein levels of activin, inhibin, follistatin, FSH and LH were measured in media of these 38 cultured pituitary adenomas. In addition, we investigated correlations between concentrations of these growth factors and hormones in NFAs and Gn-omas. RESULTS: Gn-omas were found to secrete significantly more activin A in their culture medium than PRL- and GH-producing adenomas (P < 0.05). Inhibin A and inhibin B protein levels in culture media were very low. A positive correlation between levels of activin A and FSH (r = 0.56, P < 0.005) was found, while no correlation between activin A and LH could be detected. Furthermore, levels of follistatin were positively correlated with activin A levels (r = 0.73, P < 0.0005). Comparison of the activin A:follistatin ratio with the measured FSH protein levels showed an even stronger relationship (r = 0.79, P < 0.0005). CONCLUSIONS: It is concluded that levels of activin A, follistatin and FSH in media of cultured nonfunctioning adenomas and gonadotroph adenomas are positively correlated. This suggests that these adenomas secrete FSH in response to the relatively high locally produced levels of activin A.     

The roles of inhibin and related peptides in gonadal function

Inhibin A and B are dimeric proteins capable of suppressing FSH both in vitro and in vivo. The principal form in the male is inhibin B which is produced in the testis and circulates to inhibit pituitary FSH secretion. Activin A, B and AB are dimeric proteins that share the same beta subunits with the inhibins but, in contrast, stimulate FSH secretion. Although activin A circulates, castration does not lead to a decrease in serum concentrations, indicating that the testis is not the major source of activin A. In the circulation, the activins are bound to a structurally unrelated binding protein, follistatin, that neutralizes the biological actions of these proteins. The subunits of the inhibins/activins as well as follistatin are also produced locally within the pituitary and their levels can be modulated by testosterone and gonadotrophin releasing hormone as well as by autocrine mechanisms. Consequently, the output of FSH is dependent of the balance between local processes and the circulating feedback exerted by testosterone and inhibin. There is increasing data to support the local gonadal production of not only inhibin but also activin and follistatin by both germ cells and somatic cells such as the Sertoli cells. Evidence is accumulating to support the concept that these proteins exert local regulatory mechanisms in the testis.     

Betaglycan localization in the female rat pituitary: implications for the regulation of follicle-stimulating hormone by inhibin

Activin-stimulated FSH synthesis and secretion from the pituitary gonadotrope is negatively modulated by ovarian inhibin; however, the cellular mechanism of inhibin antagonism is unknown. Inhibin and activin share a common beta-subunit through which inhibin can compete with activin for binding to the activin type II receptor and prevent activin signal transduction. Although the affinity of inhibin for binding to the activin receptor is far lower than that of activin itself, inhibin is capable of inhibiting activin-stimulated FSH synthesis and secretion even at low or equimolar concentrations. It is now known that the TGFbeta type III receptor, betaglycan, acts as an inhibin coreceptor that binds the inhibins and increases their affinity for the activin type II receptor, thereby enhancing the antagonistic effect of inhibin on activin signal transduction. Yet, despite the characterization of betaglycan is an inhibin coreceptor in several cell models in vitro, the role of this protein in the regulation of FSH in vivo has not been demonstrated. In this study we sought to understand more fully the function of betaglycan in the control of FSH release by the gonadotrope by describing betaglycan immunolocalization in the pituitary and assessing its correlation to fluctuations in FSH and inhibin throughout the rat estrous cycle. In general, betaglycan immunoreactivity was present in the anterior pituitary at all estrous cycle time points, but was confined to the membrane of gonadotropes just before and after the primary and secondary FSH surges. Importantly, betaglycan localized to the gonadotrope membrane when inhibin must rapidly reduce FSH to basal levels after the secondary FSH surge. These data indirectly support a role for betaglycan in vivo as a coreceptor that is required for inhibin-modulated FSH release from the pituitary.     

Occurrence of immunoreactive Activin/Inhibin beta(B) in thyrotropes and gonadotropes in the bullfrog pituitary: possible Paracrine/Autocrine effects of activin B on gonadotropin secretion

Occurrence of immunoreactive activin/inhibin beta(B) in the bullfrog (Rana catesbeiana) pituitary was investigated immunocytochemically by use of antibody against Xenopus activin/inhibin beta(B) subunit. Thyrotropes were demonstrated to contain activin/inhibin beta(B)-immunoreactive substances. Moreover, immunoelectron microscopy revealed that in the secretory granules of thyrotropes and, to a lesser extent, in those of gonadotropes, activin/inhibin beta(B)-immunoreactive substances were present. Based on this observation, we investigated the effect of activin B on the release of gonadotropins from dispersed anterior pituitary cells of the bullfrog. Activin B stimulated the release of not only follicle-stimulating hormone (FSH) but also luteinizing hormone (LH) dose dependently. Under the culture conditions used in this experiment, inhibin B, as well as follistatin, did not affect the basal levels of LH and FSH, but they suppressed the activin-induced release of these hormones. This is the first study on the effect of activin on pituitary hormone secretion in lower tetrapods.     

Pituitary follistatin and activin gene expression, and the testicular regulation of FSH in the adult Rhesus monkey (Macaca mulatta).

In rats, FSHbeta gene expression and FSH secretion are increased and decreased, respectively, by pituitary activin and follistatin. Because little information is available on the paracrine control of FSH secretion in the primate, follistatin and activin/inhibin beta(B) messenger RNA (mRNA) levels were measured in pituitaries of adult male rhesus monkeys 6 weeks after castration or sham surgery (n = 5/group). Follistatin mRNA was determined by quantitative RT-PCR assay using oligonucleotide primers designed to span exons 3-5 of the human follistatin gene. Activin/inhibin beta(B) mRNA levels were measured by ribonuclease protection. Orchidectomy resulted in a 100-fold increase in plasma FSH concentrations and a 60-fold rise in those of LH. In castrated monkeys, levels of mRNA encoding FSHbeta, LHbeta, alpha- subunit, and GnRH receptor (GnRH-R) were increased 21-, 2.1-, 1.7-, and 1.7-fold, respectively (P < 0.01). Levels of pituitary follistatin and activin/inhibin beta(B) mRNAs, however, were similar in castrated and intact animals. These data suggest that the paracrine control of FSH secretion in the male differs substantially in primates and rodents. Specifically, the relatively greater postcastration rise in FSHbeta gene expression and FSH secretion in the adult male monkey may result because in this species pituitary follistatin gene expression does not increase after orchidectomy, as it does in the rat.     

Proliferative phase sertoli cells display a developmentally regulated response to activin in vitro

We have used cultures of highly purified, proliferating rat Sertoli cells collected from d 3, 6, and 9 rat pups to investigate the role of activin A on Sertoli cell division. These studies demonstrate that activin A acts directly on d 6 and 9, but not d 3, Sertoli cells to induce proliferation, both alone and synergistically with FSH. In addition to stimulating proliferation, activin A induces secretion of inhibins A and B as determined by specific ELISAs. We demonstrate that the synergy between activin A and FSH is not due to local actions of secreted inhibin or follistatin. We have used real-time fluorometric RT-PCR to demonstrate that activin regulates expression of activin receptor and follistatin mRNA by Sertoli cells. Saturation binding studies using (125)I-activin A indicate that synergy between activin and FSH may be due to increased numbers of activin receptors on the Sertoli cell. Finally, we show that activin A was secreted at high levels by cultured peritubular cells but was undetectable in high purity proliferating Sertoli cell cultures, suggesting that activin A functions as a paracrine factor during postnatal testis development.     

Rat anterior pituitary folliculostellate cells are targets of interleukin-1beta and a major source of intrapituitary follistatin

Folliculostellate cells of the anterior pituitary are postulated to be an important source of factors, such as follistatin, that regulate pituitary function by intercellular communication. To gain further insight into the function of this cell type, folliculostellate cells were enriched from cultured rat anterior pituitary cells, and an immortalized cell line designated FS/D1h was established and characterized. These FS/D1h cells express S100 immunoreactivity and produce IL-6 but not pituitary hormones such as GH, ACTH, FSH, and LH. Importantly, FS/D1h cells express large amounts of follistatin mRNA and secrete the protein, as quantified indirectly by the amount of [(125)I]activin A immunoprecipitated with a follistatin antiserum. The FS/D1h cells also express alpha, betaA, and betaB inhibin/activin subunit mRNAs, but whether they produce the corresponding activins and inhibins has not been determined. The response of FS/D1h cells to agents thought to modulate folliculostellate cell function was evaluated. IL-1beta (0.005-5 nM) stimulated the secretion of follistatin and increased mRNA expression. In parallel, IL-6 secretion was stimulated. Dexamethasone, pituitary adenylate cyclase-activating polypeptide(1-27), and lipopolysaccharide but not testosterone, 12-O-tetradecanoylphorbol-13-acetate, or forskolin also increased follistatin secretion. Surprisingly, activin had no effect on follistatin mRNA levels, despite the fact that FS/D1h cells express ActRII, ActRIIB, and ALK-4 (ActRIB). Activin, on the other hand, induced Smad7 mRNA accumulation and exerted an antiproliferative effect on FS/D1h cells. Altogether, these observations support the possibility that follistatin originating from folliculostellate cells participates in mediating the effects of IL-1beta, glucocorticoids, and other agents on the response of pituitary cells to activins.     

Production and purification of recombinant human inhibin and activin.

Inhibin and activin are protein hormones with diverse physiological roles including the regulation of pituitary FSH secretion. Like other members of the transforming growth factor-beta gene family, they undergo processing from larger precursor molecules as well as assembly into functional dimers. Isolation of inhibin and activin from natural sources can only produce limited quantities of bioactive protein. To purify large-scale quantities of recombinant human inhibin and activin, we have utilized stably transfected cell lines in self-contained bioreactors to produce protein. These cells produce approximately 200 microg/ml per day total recombinant human inhibin. Conditioned cell media can be purified through column chromatography resulting in dimeric mature 32-34 kDa inhibin A and 28 kDa activin A. The purified recombinant proteins maintain their biological activity as measured by traditional in vitro assays including the regulation of FSH in rat anterior pituitary cultures and the regulation of promoter activity of the activin-responsive promoter p3TP-luc in tissue culture cells. These proteins will be valuable for future analysis of inhibin and activin function and have been distributed to the US National Hormone and Peptide Program.     

Intra-ovarian roles of activins and inhibins

Granulosa cells are the main ovarian source of inhibins, activins and activin-binding protein (follistatin) while germ (oogonia, oocytes) and somatic (theca, granulosa, luteal) cells express activin receptors, signaling components and inhibin co-receptor (betaglycan). Activins are implicated in various intra-ovarian roles including germ cell survival and primordial follicle assembly; follicle growth from preantral to mid-antral stages; suppression of thecal androgen production; promotion of granulosa cell proliferation, FSHR and CYP19A1 expression; enhancement of oocyte developmental competence; retardation of follicle luteinization and/or atresia and involvement in luteolysis. Inhibins (primarily inhibin A) are produced in greatest amounts by preovulatory follicles (and corpus luteum in primates) and suppress FSH secretion through endocrine negative feedback. Together with follistatin, inhibins act locally to oppose auto-/paracrine activin (and BMP) signaling thus modulating many of the above processes. The balance between activin-inhibin shifts during follicle development with activin signalling prevailing at earlier stages but declining as inhibin and betaglycan expression rise.     

Effects of porcine follicular fluid, inhibin-A, and activin-A on goldfish gonadotropin release in vitro.

Inhibin and activin are important reproductive regulators in mammalian species and have been demonstrated to be highly conserved in structure. The present study examines the effects of porcine follicular fluid (pFF; a crude inhibin and activin preparation) and purified porcine inhibin-A and activin-A on goldfish gonadotropin-II (GTH-II) release. In studies using primary cultures of dispersed goldfish pituitary cells in static incubation, treatments with pFF, inhibin-A, and activin-A for 10 h caused dose-dependent increase in GTH-II release. In perifusion studies using goldfish pituitary fragments, basal GTH-II release was significantly elevated after 12-h exposure to 500 micrograms/ml pFF. Furthermore, GnRH-induced GTH-II secretion was potentiated by pretreatment with pFF. When pFF was applied in the form of 5-min pulses, a rapid dose-related stimulation of GTH-II was observed. Similarly, challenges with 2-min pulses of 15, 150, and 1500 pM inhibin-A and activin-A stimulated GTH-II release by goldfish pituitary fragments in a rapid and dose-dependent manner. This acute stimulatory action of inhibin on goldfish GTH-II release was completely abolished after pretreatment with specific inhibin antibodies. The acute actions of inhibin and activin on GTH-II release are probably not due to the release of endogenous GnRH from nerve terminals in the pituitary fragments or binding to the GnRH receptors. First, a specific GnRH antagonist did not block the actions of inhibin and activin. Second, dopamine, a potent inhibitor of GnRH-stimulated GTH-II secretion in goldfish, was only partially effective in decreasing inhibin- and activin-induced GTH-II release. Third, the stimulatory effects of inhibin and GnRH on GTH-II release were additive. These lines of evidence also indicate that the mechanisms mediating inhibin and activin stimulation of goldfish GTH-II release may be somewhat different from those of GnRH. These results demonstrate that in contrast with the usual inhibitory effects of inhibin on GTH release in mammals, both inhibin and activin exert long term and acute stimulatory actions on GTH-II release in the goldfish.     

Uterine milk protein, a novel activin-binding protein, is present in ovine allantoic fluid.

Activins are pluripotent growth factors that have recently been shown to be present in placental and fetal membrane preparations. Our previous studies have identified and purified activin A from ovine amniotic and allantoic fluids. In this study, ligand blots of side fractions from the isolation of activin A from allantoic fluid suggested the presence of activin-binding proteins other than follistatin. Further purification of one of these fractions involved two sequential reverse phase HPLC steps and a Superose 12HR fractionation. SDS-PAGE revealed a single protein band of 55 kDa, which was identified by NH2-terminal sequencing as ovine uterine milk protein (UTMP), a member of the serine protease inhibitor (serpin) superfamily of proteins. Further binding studies, using ligand blot techniques and Superose 12HR fractionation in the presence of [125I]activin, demonstrated UTMP to be an activin-binding protein with a lower affinity for activin than that of follistatin. A study of the specific binding behavior of UTMP to activin, using surface plasmon resonance, revealed an apparent equilibrium dissociation constant (Kd) of 49 +/- 25 nM, compared with the follistatin-activin Kd of 379 +/- 51 pM. Similar to another activin-binding protein, alpha2-macroglobulin, UTMP was unable to neutralize the bioactivity of activin in a bioassay based on the capacity of activin to inhibit the proliferation of an MPC-11 plasmacytoma cell line. The high concentrations of this protein in uterine fluid during pregnancy and its ability to bind activin suggest that UTMP may act as a low affinity, high capacity binding protein to sequester activin in the local uterine environment.     

Inhibin and activin modulate the release of gonadotropin-releasing hormone, human chorionic gonadotropin, and progesterone from cultured human placental cells.

Although it is clear that human chorionic gonadotropin (hCG) and progesterone play fundamental roles in pregnancy, the regulation of placental production of these hormones remains to be defined. Recent evidence suggests that the human placenta expresses proteins related to inhibin (alpha beta subunits) or activin (beta beta subunits). Inhibin and activin (follicle-stimulating hormone-releasing protein) possess opposing activities in several biological systems including pituitary follicle-stimulating hormone (follitropin) secretion, erythroid differentiation, and gonadal sex-steroid production. The actions of purified inhibin and activin on hormonogenesis by primary cultures of human placental cells were studied. The addition of activin increased gonadotropin-releasing hormone (GnRH) and progesterone production and potentiated the GnRH-induced release of hCG. Inhibin by itself did not modify placental immunoreactive GnRH, hCG, and progesterone secretion but reversed the activin-induced changes. Neither inhibin nor activin influenced the release of human placental lactogen. Furthermore, transforming growth factor beta, structurally related to inhibin/activin, did not significantly influence hormone release from cultured placental cells. These results support the hypothesis that inhibin and activin may play a role in regulating the release of GnRH, hCG, and progesterone from placenta and implicate inhibin-related proteins in the endocrine physiology of human pregnancy.     

Action mechanism of inhibin in vitro--cycloheximide mimics inhibin actions on pituitary cells

The effects of inhibin on gonadotropin secretion from cultured rat anterior pituitary cells were examined by using purified porcine follicular fluid (pFF) 32 kDa inhibin. pFF 32 kDa inhibin suppressed the basal FSH secretion as well as cell content of FSH with identical ED50 values (ED50 = 1.0 ng/ml) in a dose-dependent manner, but did not alter either basal secretion or cell content of LH. On the other hand, pretreatment of the pituitary cells with pFF 32 kDa inhibin during the first 3 days resulted in suppression of subsequent LH-RH-stimulated release of both FSH and LH in a dose-dependent manner, indicating that the suppression of LH-RH-stimulated release of LH is one of the intrinsic inhibin actions on pituitary cells. The marked difference between ED50 values for FSH (ED50 = 1.1 ng/ml) and LH (ED50 = 2.5 ng/ml) in the suppression of LH-RH-stimulated release of gonadotropins, together with the fact that the total amount of LH (cell content plus released) after LH-RH stimulation remained unchanged following inhibin treatment suggests that the suppression of LH-RH-stimulated release of LH by inhibin is quite different from that of FSH regarding the action mechanism. Similarly, cycloheximide, an inhibitor of protein biosynthesis, suppressed both basal secretion and cell content of FSH with almost the same ED50 values (ED50 = 22.5 ng/ml) but did not alter either basal secretion or cell content of LH. Cycloheximide also suppressed LH-RH-stimulated release of both FSH and LH, and the ED50 values were different from each other (ED50 = 25.0 ng/ml for FSH and 60.0 ng/ml for LH suppression, respectively). Our finding that cycloheximide completely mimicked the action of inhibin on gonadotropin secretion strongly suggests that LH is quite insensitive to biosynthetic inhibition, and that preferential effects of inhibin or cycloheximide on FSH in appearance may reflect the difference between LH and FSH in susceptibility to biosynthetic inhibition.