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.     

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.     

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.     

Activin B inhibits lipolysis in 3T3-L1 adipocytes

Activin B, consisting of two inhibin βB (INHBB) subunits, is a hormone known to affect gonadal function, reproduction and fetal development. We have reported that INHBB and activin B receptors are highly expressed in adipocytes suggesting that activin B may have local effects in adipose tissue. In this study, we investigate the effect of activin B on lipolysis, measured as release of non-esterified fatty acids and free glycerol. Recombinant activin B decreased lipolysis in a concentration-dependent manner and increased intracellular triglyceride content in 3T3-L1 adipocytes. siRNA-mediated knock-down of INHBB expression increased lipolysis, and this effect was abolished by addition of recombinant activin B. In line with its inhibitory effect on lipolysis, activin B caused a down regulation of the expression of adipose triglyceride lipase and hormone sensitive lipase, key genes involved in lipolysis. In summary, we suggest that activin B is a novel adipokine that inhibits lipolysis in a paracrine or autocrine manner.     

Reconstitution and analysis of soluble inhibin and activin receptor complexes in a cell-free system

Activins and inhibins compose a heterogeneous subfamily within the transforming growth factor-beta (TGF-beta) superfamily of growth and differentiation factors with critical biological activities in embryos and adults. They signal through a heteromeric complex of type II, type I, and for inhibin, type III receptors. To characterize the affinity, specificity, and activity of these receptors (alone and in combination) for the inhibin/activin subfamily, we developed a cell-free assay system using soluble receptor-Fc fusion proteins. The soluble activin type II receptor (sActRII)-Fc fusion protein had a 7-fold higher affinity for activin A compared with sActRIIB-Fc, whereas both receptors had a marked preference for activin A over activin B. Although inhibin A and B binding was 20-fold lower compared with activin binding to either type II receptor alone, the mixture of either type II receptor with soluble TGF-beta type III receptor (TbetaRIII; betaglycan)-Fc reconstituted a soluble high affinity inhibin receptor. In contrast, mixing either soluble activin type II receptor with soluble activin type I receptors did not substantially enhance activin binding. Our results support a cooperative model of binding for the inhibin receptor (ActRII.sTbetaRIII complex) but not for activin receptors (type II + type I) and demonstrate that a complex composed of activin type II receptors and TbetaRIII is both necessary and sufficient for high affinity inhibin binding. This study also illustrates the utility of this cell-free system for investigating hypotheses of receptor complex mechanisms resulting from crystal structure analyses.     

Identification of the two types of specific receptor for activin/EDF expressed on Friend leukemia and embryonal carcinoma cells

Activin and inhibin are polypeptide factors which control the release of follicle stimulating hormone(FSH) from pituitary cells. The recent finding that erythroid differentiation factor(EDF) is identical to activin showed the multifunctional feature of this protein. To identify the specific receptor for activin, the binding of 125I-labeled activinA was investigated for a number of culture cell lines. Friends leukemia cell, which can be differentiated by activin, and embryonal carcinoma(EC) cells(PCC3, P19 and F9), were found to express 3500-20,000 per cell of activin receptors. Scatchard plot analysis of the binding data shows that the receptors on those cells could be divided into two groups with different Kd values. The Kd values of high and low affinity receptors are 0.15-0.4 nM and 1.5-3.0 nM respectively. The proportion of the number of the high and low affinity receptors was varied in each cell. Inhibin was able to compete for activin binding to both types of receptors, although the binding affinity was about 50-200 fold lower than that of activinA. Transforming growth factor-beta had no binding ability to the activin receptors.     

Activin-A, but not inhibin, regulates 17beta-hydroxysteroid dehydrogenase type 1 activity and expression in cultured rat granulosa cells

17beta-Hydroxysteroid dehydrogenase type 1 (17HSD type 1) catalyzes the reduction of estrone (E(1)) to biologically more active estradiol (E(2)). In the present study, the effect of activin, inhibin, and follistatin on 17HSD activity and 17HSD type 1 expression in cultured, unluteinized rat granulosa cells was examined. Furthermore, the effects of these hormones on 17HSD type 1 expression were compared with the expression of P450 aromatase (P450arom). Rat granulosa cells were pre-incubated in serum-free media for 3 days, followed by a 2-day treatment with activin, inhibin, follistatin and 8-Br-cAMP. Activin in increasing concentrations appeared to effect a dose-dependent increase in 17HSD activity. In addition, increasing concentrations of activin also increased 17HSD type 1 mRNA expression. Addition of 8-Br-cAMP at concentrations of 0.25 and 1.5 mmol/l together with activin significantly augmented the stimulatory effects of activin alone in the cultured cells. Neither inhibin, nor follistatin, either alone or in combination with 8-Br-cAMP, had any notable effects on 17HSD activity and 17HSD type 1 expression. Preincubation of activin with increasing concentrations of follistatin significantly diminished the stimulatory effect of activin. In the presence of follistatin, activin did not significantly increase the 8-Br-cAMP-induced 17HSD activity and 17HSD type 1 expression. The culturing of granulosa cells in the presence or the absence of inhibin or follistatin with or without 8-Br-cAMP did not alter the effect of these peptides on P450arom expression in rat granulosa cells as judged by Northern blot analysis of total RNA. However, cAMP-induced P450arom expression was enhanced by activin treatment, except when follistatin was present. This is in line with the suggested role of follistatin as an activin-binding protein, which limits the bioavailability of activin to its membrane receptors. Thus, the results support the notion of a paracrine/autocrine role of activin in follicular steroidogenesis of growing follicles.     

Activins,inhibins, and follistatins: from endocrinology to signaling. A paradigm for the new millennium

It has been 70 years since the name inhibin was used to describe a gonadal factor that negatively regulated pituitary hormone secretion. The majority of this period was required to achieve purification and definitive characterization of inhibin, an event closely followed by identification and characterization of activin and follistatin (FS). In contrast, the last 15-20 years saw a virtual explosion of information regarding the biochemistry, physiology, and biosynthesis of these proteins, as well as identification of activin receptors, and a unique mechanism for FS action-the nearly irreversible binding and neutralization of activin. Many of these discoveries have been previously summarized; therefore, this review will cover the period from the mid 1990s to present, with particular emphasis on emerging themes and recent advances. As the field has matured, recent efforts have focused more on human studies, so the endocrinology of inhibin, activin, and FS in the human is summarized first. Another area receiving significant recent attention is local actions of activin and its regulation by both FS and inhibin. Because activin and FS are produced in many tissues, we chose to focus on a few particular examples with the most extensive experimental support, the pituitary and the developing follicle, although nonreproductive actions of activin and FS are also discussed. At the cellular level, it now seems that activin acts largely as an autocrine and/or paracrine growth factor, similar to other members of the transforming growh factor beta superfamily. As we discuss in the next section, its actions are regulated extracellularly by both inhibin and FS. In the final section, intracellular mediators and modulators of activin signaling are reviewed in detail. Many of these are shared with other transforming growh factor beta superfamily members as well as unrelated molecules, and in a number of cases, their physiological relevance to activin signal propagation remains to be elucidated. Nevertheless, taken together, recent findings suggest that it may be more appropriate to consider a new paradigm for inhibin, activin, and FS in which activin signaling is regulated extracellularly by both inhibin and FS whereas a number of intracellular proteins act to modulate cellular responses to these activin signals. It is therefore the balance between activin and all of its modulators, rather than the actions of any one component, that determines the final biological outcome. As technology and model systems become more sophisticated in the next few years, it should become possible to test this concept directly to more clearly define the role of activin, inhibin, and FS in reproductive physiology.     

Activin-A, but not inhibin, regulates 17β-hydroxysteroid dehydrogenase type 1 activity and expression in cultured rat granulosa cells

17β-Hydroxysteroid dehydrogenase type 1 (17HSD type 1) catalyzes the reduction of estrone (E₁) to biologically more active estradiol (E₂). In the present study, the effect of activin, inhibin, and follistatin on 17HSD activity and 17HSD type 1 expression in cultured, unluteinized rat granulosa cells was examined. Furthermore, the effects of these hormones on 17HSD type 1 expression were compared with the expression of P450 aromatase (P450arom). Rat granulosa cells were pre-incubated in serum-free media for 3 days, followed by a 2-day treatment with activin, inhibin, follistatin and 8-Br-cAMP. Activin in increasing concentrations appeared to effect a dose-dependent increase in 17HSD activity. In addition, increasing concentrations of activin also increased 17HSD type 1 mRNA expression. Addition of 8-Br-cAMP at concentrations of 0.25 and 1.5 mmol/l together with activin significantly augmented the stimulatory effects of activin alone in the cultured cells. Neither inhibin, nor follistatin, either alone or in combination with 8-Br-cAMP, had any notable effects on 17HSD activity and 17HSD type 1 expression. Preincubation of activin with increasing concentrations of follistatin significantly diminished the stimulatory effect of activin. In the presence of follistatin, activin did not significantly increase the 8-Br-cAMP-induced 17HSD activity and 17HSD type 1 expression. The culturing of granulosa cells in the presence or the absence of inhibin or follistatin with or without 8-Br-cAMP did not alter the effect of these peptides on P450arom expression in rat granulosa cells as judged by Northern blot analysis of total RNA. However, cAMP-induced P450arom expression was enhanced by activin treatment, except when follistatin was present. This is in line with the suggested role of follistatin as an activin-binding protein, which limits the bioavailability of activin to its membrane receptors. Thus, the results support the notion of a paracrine/autocrine role of activin in follicular steroidogenesis of growing follicles.     

An activin/furin regulatory loop modulates the processing and secretion of inhibin alpha- and betaB-subunit dimers in pituitary gonadotrope cells

Of all ligands of the transforming growth factor beta superfamily, inhibins and activins are a physiologically relevant pair that are functional antagonists of each other. Activin stimulates whereas inhibin blocks follicle-stimulating hormone biosynthesis and secretion from pituitary gonadotrope cells, and together, inhibin and activin control the pituitary gonadal axis essential for normal reproductive function. Sharing a similar beta-subunit, the secretion of inhibin heterodimers (alpha/beta) or activin homodimers (beta/beta) as mature bioactive ligands depends, in part, on the proteolytic processing of precursor proteins. A short loop regulatory pathway controlling precursor processing and dimer secretion was discovered. Activin stimulates endogenous inhibin alpha- and betaB-subunit mRNA, protein, and proteolytic processing. Simultaneously, activin stimulated the proconvertase furin through a Smad2/3-dependent process. The data provide a mechanism where the regulation of furin and inhibin subunits cooperates in an important positive short feedback loop. This regulatory loop augments the secretion of bioactive mature activin B, as well as inhibin B dimers, necessary for local follicle-stimulating hormone beta regulation.     

Local roles of TGF-beta superfamily members in the control of ovarian follicle development

Members of the transforming growth factor-beta (TGF-beta) superfamily have wide-ranging influences on many tissue and organ systems including the ovary. Two recently discovered TGF-beta superfamily members, growth/differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15; also designated as GDF-9B) are expressed in an oocyte-specific manner from a very early stage and play a key role in promoting follicle growth beyond the primary stage. Follicle growth to the small antral stage does not require gonadotrophins but appears to be driven by local autocrine/paracrine signals from both somatic cell types (granulosa and theca) and from the oocyte. TGF-beta superfamily members expressed by follicular cells and implicated in this phase of follicle development include TGF-beta, activin, GDF-9/9B and several BMPs. Acquisition of follicle-stimulating hormone (FSH) responsiveness is a pre-requisite for growth beyond the small antral stage and evidence indicates an autocrine role for granulosa-derived activin in promoting granulosa cell proliferation, FSH receptor expression and aromatase activity. Indeed, some of the effects of FSH on granulosa cells may be mediated by endogenous activin. At the same time, activin may act on theca cells to attenuate luteinizing hormone (LH)-dependent androgen production in small to medium-size antral follicles. Dominant follicle selection appears to depend on differential FSH sensitivity amongst a growing cohort of small antral follicles. Activin may contribute to this selection process by sensitizing those follicles with the highest "activin tone" to FSH. Production of inhibin, like oestradiol, increases in selected dominant follicles, in an FSH- and insulin-like growth factor-dependent manner and may exert a paracrine action on theca cells to upregulate LH-induced secretion of androgen, an essential requirement for further oestradiol secretion by the pre-ovulatory follicle. Like activin, BMP-4 and -7 (mostly from theca), and BMP-6 (mostly from oocyte), can enhance oestradiol and inhibin secretion by bovine granulosa cells while suppressing progesterone secretion; this suggests a functional role in delaying follicle luteinization and/or atresia. Follistatin, on the other hand, may favor luteinization and/or atresia by bio-neutralizing intrafollicular activin and BMPs. Activin receptors are expressed by the oocyte and activin may have a further intrafollicular role in the terminal stages of follicle differentiation to promote oocyte maturation and developmental competence. In a reciprocal manner, oocyte-derived GDF-9/9B may act on the surrounding cumulus granulosa cells to attenuate oestradiol output and promote progesterone and hyaluronic acid production, mucification and cumulus expansion.     

Spatial expression patterns of activin and its signaling system in the zebrafish ovarian follicle: evidence for paracrine action of activin on the oocytes

We have previously demonstrated that activin is likely an ovarian mediator of pituitary gonadotropin(s) and local epidermal growth factor in their stimulating oocyte maturation and maturational competence in the zebrafish. However, the downstream events controlled by activin remain unknown. One possible mechanism is that activin may directly work on the oocytes to promote the development of oocyte maturational competence. To substantiate this hypothesis, we performed the present study to demonstrate the expression of the activin system in different compartments of zebrafish follicles, namely, the follicle cells and oocytes. The proteins examined include activin subunits (betaA and betaB), activin-binding protein (follistatin), activin type II receptors (type IIA and IIB), the type I activin receptor-like kinases (ALK1-like, ALK2-like, and ALK4-like), and the intracellular activin signaling molecules (Smad2, Smad3, Smad4, and Smad7). The results showed that the entire activin signaling system is expressed by the full-grown immature zebrafish oocytes ( approximately 0.65 mm in diameter), including ALK4-like (ActRIB), ALK2-like (ActRIA), ActRIIA, ActRIIB, Smad2, Smad3, Smad4, and Smad7, therefore supporting our hypothesis that the oocytes are one of the direct targets of activin actions in the zebrafish ovary. In contrast, activin itself (betaA and betaB) and ALK1-like type I receptor are predominantly expressed in the follicle cells surrounding the oocytes. Interestingly, although follistatin is expressed in both the follicle cells and oocytes, its level of expression is significantly higher in the oocytes than the follicle cells, implying that follistatin may serve as a signal from the oocytes to modulate the activity of activin produced by the follicle cells. Taken together, the present study provides convincing evidence that although all members of the activin system are expressed in the whole follicle, they exhibit distinct spatial patterns of expression among different compartments of the follicle. It is likely that activin works directly on the oocytes in a paracrine manner to promote oocyte maturation and maturational competence. On the other hand, instead of being controlled passively by the follicle cells, the oocytes may actively participate in the regulation of follicle development by releasing various modulating molecules such as follistatin.