Structural basis for a functional antagonist in the transforming growth factor beta superfamily

Within the transforming growth factor beta superfamily, the agonist-antagonist relationship between activin and inhibin is unique and critical to integrated reproductive function. Activin acts in the pituitary to stimulate follicle-stimulating hormone, and is antagonized by endocrine acting, gonadally derived inhibin. We have undertaken a mutational analysis of the activin betaA subunit to determine the precise structural aspects that contribute to inhibin antagonism of activin. By substituting specific amino acid residues in the activin betaA subunit with similarly aligned amino acids from the alpha subunit, we have pinpointed the residues required for activin receptor binding and activity, as well as for inhibin antagonism of activin through its receptors. Additionally, we have identified an activin mutant with a higher affinity for the activin type I receptor that provides structural evidence for the evolution of ligand-receptor interactions within the transforming growth factor beta superfamily.     

Expression and processing of the activin-A/erythroid differentiation factor precursor: a member of the transforming growth factor-beta superfamily

The biosynthesis and intracellular processing of the polypeptide precursor of the beta A-chain of the fertility hormone inhibin were assessed by infecting a wide spectrum of cell types with a recombinant vaccinia virus. Most cell lines, including follicular granulosa cells, secrete both prohormone and mature hormone as homodimers (activin) composed of disulfide-linked subunits of 54 kDa (proactivin-A) and 14 kDa (activin-A), respectively, and a small amount of prohormone-mature hormone heterodimers. Mature activin is secreted from mouse pituitary cells (AtT-20), while pig kidney cells [PK(15)] secrete mostly proactivin. More prohormone is secreted in the presence of NH4Cl, suggesting that prohormone processing is facilitated by low pH. Proactivin-A is not a ligand for the mannose-6-phosphate/insulin growth factor-II receptor. The recombinant activin stimulates FSH release from pituitary cells and differentiates erythroleukemia cell lines in vitro.     

Transforming growth factor-beta and activin inhibit basal secretion of prolactin in a pituitary monolayer culture system

Incubations of rat anterior pituitary cells with transforming growth factor (TGF)-beta 1 for 48 hr suppressed the secretion of basal prolactin (PRL) in a dose-dependent manner (ED50, 100 pg/ml). Activin, a gonadal hormone processing cysteine distribution similar to TGF beta, also suppressed basal PRL secretion, but it was less effective (ED50, 4 mg/ml). Treatment with TGF beta 1 significantly suppressed basal PRL secretion from the pituitary after 24 hr and up to 72 hr of incubation. TGF beta 1 also inhibited thyrotropin-releasing hormone-mediated PRL secretion and activin inhibited thyrotropin-releasing hormone-mediated PRL secretion slightly, but significantly. In addition, we also measured the secretion of growth hormone by cultured pituitary cells treated with TGF beta 1 or activin for 24 to 72 hr. TGF beta 1 and activin showed an opposite effect on growth hormone secretion; TGF beta stimulated and activin inhibited basal secretion of growth hormone. These results suggest that TGF beta 1 is a potent inhibitor of basal secretion of PRL by the pituitary, and both TGF beta 1 and activin play a multifunctional role in basal secretion of pituitary hormones.     

Role of PCSK5 expression in mouse ovarian follicle development: identification of the inhibin α- and β-subunits as candidate substrates

Inhibin and activin are essential dimeric glycoproteins belonging to the transforming growth factor-beta (TGFβ) superfamily. Inhibin is a heterodimer of α- and β-subunits, whereas activin is a homodimer of β-subunits. Production of inhibin is regulated during the reproductive cycle and requires the processing of pro-ligands to produce mature hormone. Furin is a subtilisin-like proprotein convertase (proconvertase) that activates precursor proteins by cleavage at basic sites during their transit through the secretory pathway and/or at the cell surface. We hypothesized that furin-like proconvertases are central regulators of inhibin α- and β-subunit processing within the ovary. We analyzed the expression of the proconvertases furin, PCSK5, PCSK6, and PCSK7 in the developing mouse ovary by real-time quantitative RT-PCR. The data showed that proconvertase enzymes are temporally expressed in ovarian cells. With the transition from two-layer secondary to pre-antral follicle, only PCSK5 mRNA was significantly elevated. Activin A selectively enhanced expression of PCSK5 mRNA and decreased expression of furin and PCSK6 in cultured two-layer secondary follicles. Inhibition of proconvertase enzyme activity by dec-RVKR-chloromethylketone (CMK), a highly specific and potent competitive inhibitor of subtilisin-like proconvertases, significantly impeded both inhibin α- and β-subunit maturation in murine granulosa cells. Overexpression of PC5/6 in furin-deficient cells led to increased inhibin α- and β(B)-subunit maturation. Our data support the role of proconvertase PCSK5 in the processing of ovarian inhibin subunits during folliculogenesis and suggest that this enzyme may be an important regulator of inhibin and activin bioavailability.     

Role of inhibin and activin in the modulation of gonadotropin- and steroid-induced oocyte maturation in the teleost Fundulus heteroclitus

Background  

Activin and inhibin are glycoproteins structurally related to the transforming growth factor-beta superfamily. These peptides were first described as factors that regulate the follicle-stimulating hormone (FSH) at the pituitary level. The possible role of inhibin and activin, at the ovarian level, in mediating the stimulatory actions of a Fundulus pituitary extract (FPE) and 17alpha,20beta-dihydroprogesterone (DHP) on oocyte maturation was investigated in this study.     

The measurement of activin/EDF in mouse serum: evidence for extragonadal production.

Many studies have shown that activin/EDF mediates local physiological events at various sites. In this study, the authors confirmed the presence of activin in mouse serum by high performance liquid chromatography (HPLC) monitored by a specific bioassay. The retention time of the active fraction in HPLC was identical to that of authentic activin A, and the activity was neutralized by follistatin. That the serum activin levels in ovariectomized and aged mice were decreased suggests that the serum activin was generated partly by ovary (35%), but also by extragonadal organs. Activin and inhibin are structurally closely related, and both are involved in many physiological processes including control of follicle stimulating hormone secretion by the pituitary. The regulation of serum activin, however, appeared to differ from that of inhibin.     

Neuroendocrine control of FSH secretion: IV. Hypothalamic control of pituitary FSH-regulatory proteins and their relationship to changes in FSH synthesis and secretion

The current dogma is that the differential regulation of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) synthesis and secretion is modulated by gonadotropin-releasing hormone (GnRH) pulse frequency and by changes in inhibins, activins, and follistatins both at the pituitary and at the peripheral level. To date no studies have looked at the overlapping function of these regulators in a combined setting. We tested the hypothesis that changes in GnRH pulse frequency alter the relative abundance of these regulators at the pituitary and peripheral levels in a manner consistent with changes in pituitary and circulating concentrations of FSH; that is, an increase in FSH will be accompanied by increased stimulatory input (activin) and/or reduced follistatin and inhibin. Ovariectomized ewes were subjected to a combination hypothalamic pituitary disconnection (HPD)-hypophyseal portal blood collection procedure. Hypophyseal portal and jugular blood samples were collected for a 6-h period from non-HPD ewes, HPD ewes, or HPD ewes administered GnRH hourly or every 3 h for 4 days. In the absence of endogenous hypothalamic and ovarian hormones that regulate gonadotropin secretion, 3-hourly pulses of GnRH increased pituitary content of FSH more than hourly GnRH, although these differences were not evident in the peripheral circulation. The results failed to support the hypothesis in that the preferential increase of pituitary content of FSH by the lower GnRH pulse frequency could be explained by changes in the pituitary content of inhibin A, follistatin, or activin B. Perhaps the effects of GnRH pulse frequency on FSH is due to changes in the balance of free versus bound amounts of these FSH regulatory proteins or to the involvement of other regulators not monitored in this study.     

Inhibin, activin, and follistatin: regulation of follicle-stimulating hormone messenger ribonucleic acid levels

Primary pituitary cell cultures derived from adult male rats were used to explore the direct effects of purified porcine inhibin and follistatin, and recombinant human activin A on FSH beta, as well as LH beta and alpha-subunit mRNA levels. Subunit mRNAs were determined by blot hybridization using alpha, LH beta, and FSH beta cDNA and genomic fragments. Treatment with inhibin for 72 h significantly suppressed alpha and FSH beta mRNA levels with parallel changes in FSH secretion. No change in LH beta mRNA levels was observed. A decrease in FSH beta mRNA to undetectable levels was seen 4 h after inhibin administration. Recombinant human Activin A caused dose-dependent and parallel increases in FSH beta mRNA levels and FSH secretion. This increase was evident at 4 h after activin administration and maintained at longer times. alpha and LH beta mRNA levels remained unchanged. Follistatin addition to cultures for 72 h significantly reduced FSH beta mRNA levels. In a time-course experiment, a reduction in FSH beta mRNA to undetectable levels was observed 24 h after follistatin administration. There were no changes in alpha or LH beta mRNA levels. These data demonstrate that the actions of these gonadal peptides on FSH secretion may be accounted for, at least in part at the level of biosynthesis, by reductions in FSH beta mRNA levels directly at the level of the anterior pituitary gland.     

Identification of a nuclear localization signal in activin/inhibin betaA subunit; intranuclear betaA in rat spermatogenic cells

Activin is a dimeric glycoprotein hormone that was initially characterized by its ability to stimulate pituitary FSH secretion and was subsequently recognized as a growth factor with diverse biological functions in a large variety of tissues. In the testis, activin has been implicated in the auto/paracrine regulation of spermatogenesis through its cognate cell membrane receptors on Sertoli and germ cells. In this study we provide evidence for intranuclear activin/inhibin betaA subunit and show its distribution in the rat seminiferous epithelium. We have shown by transient expression in HeLa cells of beta-galactosidase fusion proteins that the betaA subunit precursor contains a functional nuclear localization signal within the lysine-rich sequence corresponding to amino acids 231-244. In all stages of the rat seminiferous epithelial cycle, an intense immunohistochemical staining of nuclear betaA was demonstrated in intermediate or type B spermatogonia or primary spermatocytes in their initial stages of the first meiotic prophase, as well as in pachytene spermatocytes and elongating spermatids primarily in stages IX-XII. In some pachytene spermatocytes, the pattern of betaA immunoreactivity was consistent with the characteristic distribution of pachytene chromosomes. In the nuclei of round spermatids, betaA immunoreactivity was less intense, and in late spermatids it was localized in the residual cytoplasm, suggesting disposal of betaA before spermatozoal maturation. Immunoblot analysis of a protein extract from isolated testicular nuclei revealed a nuclear betaA species with a molecular mass of approximately 24 kDa, which is more than 1.5 times that of the mature activin betaA subunit present in activin dimers. These results suggest that activin/inhibin betaA may elicit its biological functions through two parallel signal transduction pathways, one involving the dimeric molecule and cell surface receptors and the other an alternately processed betaA sequence acting directly within the nucleus. According to our immunohistochemical data, betaA may play a significant role in the regulation of nuclear functions during meiosis and spermiogenesis.     

Normal reproductive function in InhBP/p120-deficient mice

The inhibins are gonadal transforming growth factor beta superfamily protein hormones that suppress pituitary follicle-stimulating hormone (FSH) synthesis. Recently, betaglycan and inhibin binding protein (InhBP/p120, also known as the product of immunoglobulin superfamily gene 1 [IGSF1]) were identified as candidate inhibin coreceptors, shedding light on the molecular basis of how inhibins may affect target cells. Activins, which are structurally related to the inhibins, act within the pituitary to stimulate FSH production. Betaglycan increases the affinity of inhibins for the activin type IIA (ACVR2) receptor, thereby blocking activin binding and signaling through this receptor. InhBP/p120 may not directly bind inhibins but may interact with the activin type IB receptor, ALK4, and participate in inhibin B's antagonism of activin signaling. To better understand the in vivo functions of InhBP/p120, we characterized the InhBP/p120 mRNAs and gene in mice and generated InhBP/p120 mutant mice by gene targeting in embryonic stem cells. InhBP/p120 mutant male and female mice were viable and fertile. Moreover, they showed no alterations in FSH synthesis or secretion or in ovarian or testicular function. These data contribute to a growing body of evidence indicating that InhBP/p120 does not play an essential role in inhibin biology.     

Activin as a cell differentiation factor

Activin, originally discovered as a polypeptide hormone that is capable of stimulating follicle-stimulating hormone secretion from pituitary cells in vitro, has recently been found to have a much wider range of biological activities. There are a number of reports of activin action as a cell differentiation factor on various types of cells rather than as a modulator of hormone secretion, as predicted initially, based on its structural similarity to transforming growth factor-β. Studies of the distribution of activin and its receptor in a variety of tissues and its wide-ranging actions clearly illustrates its multifunctional properties. In particular, activin has been shown to be a potential regulator of early development of Xenopus laevis. Observation of activin effect in embryogenesis is of general importance to our understanding of the role of the family of growth factors in developmental processes.     

Roles of the activin regulatory system in fish reproduction

Activin (betaAbetaA, betaAbetaB, and betaBbetaB) is a dimeric growth factor with diverse biological activities in vertebrate reproduction. Activin exerts its actions by binding to its specific type II and type I receptors. The activity of activin is regulated by follistatin, its binding protein, and the antagonists inhibin and antivin. All major components of the activin-inhibin-follistatin system have been identified in fish except the alpha subunit of inhibin. Using goldfish as a model, we have demonstrated that activin is expressed in the pituitary and the recombinant goldfish activin B has novel inverse effects on the expression of GTH beta subunits. Activin increases the mRNA level of GTH-Ibeta while significantly suppressing the expression of GTH-IIbeta. We have also demonstrated the expression of activin and its receptors in the goldfish and zebrafish ovary. Using an in vitro ovarian follicle incubation as the system, we have investigated the involvement of the activin system in the process of final oocyte maturation. Our evidence clearly indicates that activin has potent effect of promoting final oocyte maturation, and that it may play a role in mediating the stimulatory effect of pituitary gonadotropin in the event of oocyte maturation.     

N-linked oligosaccharides direct the differential assembly and secretion of inhibin alpha- and betaA-subunit dimers

The biosynthetic pathway governing inhibin heterodimer (alpha/beta) and activin homodimer (beta/beta) assembly and secretion from ovarian granulosa cells is not fully understood. Here, we examined the role of inhibin subunit glycosylation in the assembly and secretion of mature inhibin A and activin A. Inhibition of subunit glycosylation by tunicamycin treatment of alpha- and beta(A)-expressing CHO cell lines reduced inhibin but not activin secretion. Dimeric inhibin A is preferentially secreted from parental isogenic wild-type (wt) cell lines (alpha(wt)beta(wt)). Mutation of a single glycosylation site at asparagine 268 (alpha(Delta268)beta(wt)) reduces inhibin secretion by 78% and permits beta/beta assembly and secretion. Conversely, gain of a glycosylation (GOG) site in the analogous region of the beta(A)-subunit (alpha(wt)beta(GOG327)) enhances inhibin A secretion. The present study demonstrates that N-linked glycan sites direct heterodimer vs. homodimer assembly, and prevention of glycosylation abrogates inhibin secretion. These data support a definitive role for site-specific N-glycosylation in governing inhibin/activin dimer assembly and secretion.     

Steroid-protein interaction in human placenta

Human placenta produces a large variety of bioactive substances with endocrine and neural competence: pituitary and gonadal hormones, hypothalamic-like releasing or inhibiting hormones, growth factors, cytokines and neuropeptides. The most recent findings indicate that locally produced hormones regulate the secretion of other placental hormones supporting a paracrine/autocrine regulation. In placental endocrinology, a particular relevance is played by steroid hormones. In fact, a specific gonadotropin-releasing hormone (GnRH)-human chorionic gonadotropin (hCG) regulation of placental steroidogenesis has been proposed as a placental internal regulatory system acting on steroids production from human placenta. In addition, activin and inhibin have been proposed as further regulatory substances of the synthesis and secretion of steroids; the addition of activin A to placental culture augments GnRH, hCG and progesterone, and this effect can be significantly reduced by the addition of inhibins. Finally, a steroid-steroid interaction is suggested by the evidence that placental estrogen has a positive role in the regulation of progesterone biosynthesis. Other steroid-protein interactions have been observed in human placenta. In fact, recent data indicate that progesterone inhibits placental corticotropin-releasing factor (CRF) and estrogens act on placental conversion of cortisol to cortisone, activating cortisol secretion by the fetal adrenal and enhancing fetal adrenal function with advancing gestation.     

Inhibition of somatotroph growth and growth hormone biosynthesis by activin in vitro

Activin-A, a homodimeric protein composed of two inhibin beta A-subunits, was first isolated from gonadal fluids based upon its ability to stimulate FSH secretion and biosynthesis, but was also observed to suppress GH secretion. The present report describes the effects of activin on the biosynthesis of GH and the proliferation of pituitary somatotrophs. In pituitary cells cultured in the presence of 0.7 nM activin for 3 days, GH secretion was decreased by 50% compared to the control value. Inhibition of GH biosynthesis, measured by quantitative immunoprecipitation of [35S]methionine-labeled cells, could be observed after 24 h of activin treatment, and maximal (70%) inhibition of GH biosynthesis was observed after 3 days. Activin inhibited basal as well as GH-releasing factor (GRF)-, glucocorticoid-, and thyroid hormone-stimulated GH biosynthesis. Inhibin, which is known to reverse the effect of activin on FSH secretion, did not reverse the effect of activin on GH biosynthesis. Treatment of somatotrophs with activin for 3 days completely inhibited the growth-promoting effect of GRF on somatotrophs. However, no effect of activin on GRF-stimulated expression of the c-fos protooncogene was observed. These data demonstrate that activin, in addition to its stimulatory effect on FSH secretion, is able to inhibit both expression of GH and growth of somatotropic cells.     

Rat inhibin: molecular cloning of alpha- and beta-subunit complementary deoxyribonucleic acids and expression in the ovary

Inhibin is a gonadal protein hormone that suppresses the secretion of FSH from pituitary gonadotrophs. It has previously been characterized as a heterodimer of two dissimilar subunits (alpha, 18 kilodaltons and beta, 14 kilodaltons) the smaller of which exists in two forms (beta A and beta B) and can form dimers that stimulate the secretion of FSH. In the present work, cDNA clones encoding the inhibin alpha- and beta A-subunits have been isolated from rat ovary and characterized. The alpha-inhibin cDNA predicts a precursor protein of 366 amino acids containing the 133 amino acid mature alpha-subunit at its COOH-terminus. The beta A-inhibin cDNA predicts a precursor protein of 424 amino acids containing the 116 amino acid beta A-subunit at its COOH-terminus. Analysis of rat ovarian RNA indicates that alpha-inhibin mRNA levels are stimulated by PMSG treatment in vivo. In cultured granulosa cells, FSH also stimulates alpha-inhibin mRNA, and the FSH effect is suppressed by cotreatment with GnRH. Hybridization in situ to rat ovarian tissue demonstrates that both the alpha-inhibin and beta A-inhibin mRNAs are specifically expressed in granulosa cells of the developing follicles.     

The isolation and physiology of inhibin and related proteins

Inhibin, a glycoprotein that preferentially suppresses follicle-stimulating hormone (FSH) secretion, has been isolated from follicular fluid as a heterodimer of two dissimilar subunits linked by disulphide bonds. The larger subunit is termed alpha and the smaller is designated beta. Two forms of inhibin termed A and B have been isolated, the differences being due to variations in the amino acid sequence of the beta-subunit; Inhibin A consists of alpha-beta and Inhibin B of alpha-beta B. Dimers of the beta-subunit, termed activins, have also been found in follicular fluid; these stimulate pituitary FSH secretion. Inhibin is produced in the female by the granulosa cell and corpus luteum under the control of FSH and luteinizing hormone (LH), respectively. The levels in serum rise to peak at mid-cycle and in the mid-luteal phase of the human menstrual cycle, and decline prior to menstruation. In pregnancy, the late-luteal phase decline in inhibin does not occur and the levels increase slowly. Studies suggest that the levels in pregnancy arise from an embryonic source, particularly the placenta. In the male, inhibin is produced by the Sertoli cells under the control of FSH by mechanisms involving cyclic adenosine 3', 5'-monophosphate. Testosterone exerts a minor inhibitory control at supraphysiological levels (10(-5) M), but human chorionic gonadotropin stimulation results paradoxically in a rise in serum inhibin levels. Disruption of spermatogenesis in the rat by cryptorchidism, heat treatment, or efferent duct ligation results in a decline in inhibin levels and a rise in FSH levels, findings consistent with the negative feedback action of inhibin on FSH secretion. As well as their roles in the reproductive system, inhibin and activin have more widespread actions in the haemopoietic, immune and nervous systems as evidenced by the finding of mRNA for its subunits in a range of tissues. Other studies have shown actions on erythroid differentiation and on mitotic activity in thymocytes. These actions suggest that inhibin and activin may function as growth factors as well as regulators of FSH.